CN114717237B

CN114717237B - EP6 promoter and related biological material and application thereof

Info

Publication number: CN114717237B
Application number: CN202210650799.3A
Authority: CN
Inventors: 魏爱英; 孟刚; 贾慧萍; 米杰; 杨立鹏; 苏厚波; 田斌
Original assignee: Beijing Zhongke Yipin Biotechnology Co ltd
Current assignee: Beijing Zhongke Yipin Biotechnology Co ltd
Priority date: 2022-06-10
Filing date: 2022-06-10
Publication date: 2022-08-19
Anticipated expiration: 2042-06-10
Also published as: WO2023236634A1; CN114717237A

Abstract

The invention discloses an EP6 promoter, a related biological material and application thereof. The invention discloses an EP6 promoter, the sequence of which is SEQ ID No.3 in a sequence table. According to the invention, the EP6 promoter is used for starting the gene in the corresponding amino acid synthesis pathway to obtain the corresponding expression cassette, and then the expression cassette is used as an exogenous strong promoter and integrated into the bacteria with high amino acid yield to replace the original promoter of the key gene for amino acid synthesis, so that the yield of the corresponding amino acid is improved. The EP6 promoter has high promoter activity, can be used for producing amino acid, and has a great application prospect.

Description

EP6 promoter, related biological material thereof and application

Technical Field

The invention relates to the technical field of biology, in particular to an EP6 promoter, a related biological material and application thereof.

Background

At present, the level of industrially producing amino acid is greatly improved, but how to further improve the yield of amino acid on the basis of the higher level needs to fully consider a carbon source required by the growth of bacteria producing amino acid and how to maximally enter an amino acid synthesis pathway in the later stage of fermentation. To solve this problem, in the case of bacteria producing a certain amino acid, promoters of different strengths need to be applied to different genes of the corresponding amino acid metabolic pathway to achieve the balance of the amino acid metabolic pathway and bacterial growth.

Disclosure of Invention

The object of the present invention is to provide an EP6 promoter with which amino acids can be produced using the EP6 promoter.

The EP6 promoter provided by the invention has a sequence of SEQ ID No.3 in a sequence table.

The invention also provides a biological material related to the EP6 promoter, wherein the biological material is any one of the following B1) to B7):

B1) an expression cassette comprising the EP6 promoter;

B2) a recombinant vector comprising the EP6 promoter;

B3) a recombinant vector comprising the expression cassette of B1);

B4) a recombinant microorganism comprising an EP6 promoter;

B5) a recombinant microorganism comprising the expression cassette of B1);

B6) a recombinant microorganism containing the recombinant vector of B2);

B7) a recombinant microorganism comprising the recombinant vector of B3).

In the above biological materials, the expression cassette containing the EP6 promoter described in B1) refers to a DNA capable of driving the expression of a target gene by the EP6 promoter in a host cell, and the DNA may include not only the target gene but also a terminator for terminating the transcription of the target gene. Further, the expression cassette may also include an enhancer sequence.

The recombinant vector containing the EP6 promoter can be constructed using an existing expression vector.

In the above biological material, the vector may be a plasmid, a cosmid, a phage, or a viral vector.

In the above biological material, the microorganism may be yeast, bacteria, algae or fungi. Wherein the bacteria can be Corynebacterium glutamicum (C.) (Corynebacterium glutamicum) Brevibacterium lactofermentum, Brevibacterium flavum: (B) ((B))brevibacterium flavum) Beijing Corynebacterium (C. Beijing)Corynebacterium pekinense) Brevibacterium ammoniavorum, Corynebacterium crenatum or Pantoea bacterium: (Pantoea）。

The invention also provides an application of the EP6 promoter as a promoter.

The invention also provides the use of the EP6 promoter or the biological material for the production of amino acids.

In the above application, the amino acid may be lysine, glutamic acid or valine.

The EP6 promoter of the present invention can be used to produce a variety of products including, but not limited to, lysine, glutamic acid and valine in the examples, glycine, alanine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, arginine, histidine, shikimic acid, protocatechuic acid, succinic acid, alpha ketoglutaric acid, citric acid, ornithine, citrulline, and the like. In the production of various products, the production of a desired product can be achieved by placing the EP6 promoter of the present invention upstream of a gene in the pathway for synthesis of a desired product and allowing the EP6 promoter to drive the synthesis of the gene in the pathway for synthesis of the desired product.

The present invention also provides a method for producing an amino acid, the method comprising: introducing an EP6 promoter into a biological cell capable of synthesizing a target amino acid, and driving the expression of genes in a target amino acid synthesis pathway in the biological cell by the EP6 promoter to obtain a recombinant biological cell; and culturing the recombinant biological cells to obtain the target amino acid.

In the above method, the biological cell may be a yeast, a bacterium, an algae, a fungus, a plant cell or an animal cell capable of synthesizing the desired amino acid.

The biological cell is any biological cell capable of synthesizing the target amino acid.

The bacterium can be Corynebacterium glutamicum (C.), (Corynebacterium glutamicum) Brevibacterium lactofermentum, Brevibacterium flavum: (brevibacterium flavum) Beijing Corynebacterium (C. Beijing)Corynebacterium pekinense) Brevibacterium ammoniavorum, Corynebacterium crenatum or Pantoea bacterium: (Pantoea）。

In one embodiment of the present invention, the desired amino acid is lysine, and the bacterium is Corynebacterium glutamicum: (C)Corynebacterium glutamicum）CGMCC No.12856。

Bacteria for producing lysine using the promoter of the present invention include, but are not limited to, Corynebacterium glutamicum: (C.), (Corynebacterium glutamicum) CGMCC No. 12856. The present invention can place the EP6 promoter of the present invention upstream of genes in the lysine synthesis pathway of these bacteria, so that the EP6 promoter of the present invention can synthesize lysine by driving expression of genes in the lysine synthesis pathway in these bacteria.

In one embodiment of the present invention, the desired amino acid is glutamic acid, and the bacterium is Corynebacterium glutamicum: (C)Corynebacterium glutamicum）CGMCC No. 21220。

Bacteria for producing glutamic acid using the promoter of the present invention include, but are not limited to, Corynebacterium glutamicum (C.), (Corynebacterium glutamicum) CGMCC number 21220. The present invention can place the EP6 promoter of the present invention upstream of genes in the glutamic acid synthesis pathway of these bacteria, so that the EP6 promoter of the present invention can synthesize glutamic acid by driving expression of genes in the glutamic acid synthesis pathway in these bacteria.

In one embodiment of the present invention, the desired amino acid is valine and the bacterium is Corynebacterium glutamicum: (C)Corynebacterium glutamicum）CGMCC No.21260。

Bacteria for producing valine using the promoter of the present invention include, but are not limited to, Corynebacterium glutamicum ((C.))Corynebacterium glutamicum) CGMCC No. 21260. The present invention can place the EP6 promoter of the present invention in the valine synthetic pathway of these bacteriaUpstream, the EP6 promoter of the present invention is made capable of synthesizing valine by driving the expression of genes in the valine synthetic pathway in these bacteria.

In the above method, the desired amino acid may be lysine, glutamic acid or valine.

The EP6 promoter of the present invention can be used to produce a variety of products including, but not limited to, lysine, glutamic acid and valine in the examples, glycine, alanine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, arginine, histidine, shikimic acid, protocatechuic acid, succinic acid, alpha ketoglutaric acid, citric acid, ornithine, citrulline, and the like. When producing various target products, the production of the target product can be achieved by placing the EP6 promoter of the present invention upstream of the gene in the synthesis pathway of the target product and allowing the EP6 promoter to drive the synthesis of the gene in the synthesis pathway of the target product.

In one embodiment of the present invention, the desired amino acid is lysine, and the bacterium is Corynebacterium glutamicum: (C)Corynebacterium glutamicum) CGMCC No.12856, the gene in the lysine synthesis pathway is lysA gene (position 149-1486 of SEQ ID No. 4).

The recombinant biological cells are obtained by replacing the EP6 promoter with Corynebacterium glutamicum (C.glutamicum)Corynebacterium glutamicum) The lysA gene original promoter in CGMCC No. 12856.

In one embodiment of the present invention, the desired amino acid is glutamic acid, and the bacterium is Corynebacterium glutamicum: (C)Corynebacterium glutamicum) CGMCC No.21220, the gene in the synthesis pathway of glutamic acid is BBD29_14295 gene (37-1161 th site of SEQ ID No. 11).

The recombinant biological cells are obtained by replacing the EP6 promoter with Corynebacterium glutamicum (C.glutamicum)Corynebacterium glutamicum) The CGMCC No.21220, BBD 29-14295 gene original promoter.

In one embodiment of the invention, the target amino acid is valineThe bacterium is Corynebacterium glutamicum (C.) (Corynebacterium glutamicum) CGMCC No.21260, the gene in the valine synthetic pathway is ilvC gene (position 180-1196 of SEQ ID No. 18).

The recombinant biological cells are obtained by replacing the EP6 promoter with Corynebacterium glutamicum (C.glutamicum)Corynebacterium glutamicum) The ilvC gene original promoter in CGMCC No. 21260.

For bacteria producing amino acid, the EP6 promoter is used to start the gene in the corresponding amino acid synthesizing path to obtain the corresponding expression cassette, and the expression cassette is used as exogenous strong promoter integrated into the bacteria producing amino acid in high yield to replace the original promoter of the key gene for synthesizing amino acid, so as to raise the yield of corresponding amino acid. The EP6 promoter has high promoter activity, can be used for producing amino acid, and has a great application prospect.

The present invention is described in further detail below with reference to specific embodiments, and the examples are given only for illustrating the present invention and not for limiting the scope of the present invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

Biological material preservation instructions.

And (3) classification and naming: corynebacterium glutamicum (C)Corynebacterium glutamicum）。

The strain number is as follows: YPGLU 001.

Name of the depository: china general microbiological culture Collection center (CGMCC).

The preservation unit is abbreviated as: CGMCC.

The address of the depository: west road No.1, north chen, chaoyang district, beijing, zip code: 100101.

the preservation date is as follows: year 2020, 11, 23.

Registration number of the preservation center: CGMCC No. 21220.

Biological material preservation description.

And (3) classification and naming: corynebacterium glutamicum (C.) (Corynebacterium glutamicum）。

The strain number is as follows: YPFV 1.

The name of the depository: china general microbiological culture Collection center.

The preservation unit is abbreviated as: CGMCC.

The address of the depository: west road No.1 hospital No.3, beijing, chaoyang district, zip code: 100101.

the preservation date is as follows: year 2020, 11, 30 months.

Registration number of the preservation center: CGMCC No. 21260.

Biological material preservation instructions.

The strain number is as follows: YP 097158.

The preservation unit is abbreviated as: CGMCC.

the preservation date is as follows: 2016, 8, 16 months.

The registration number of the collection center: CGMCC No. 12856.

Drawings

FIG. 1 shows the results of the detection of the promoter activities of EP6 and yfjB. PEP6 is EP6 promoter.

Detailed Description

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. In the quantitative tests in the following examples, three replicates were set up and the results averaged. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA/RNA.

pEC-H10-mCherry vector: the preparation method described in the chinese patent application No. 202110256579.8 (publication No. CN 112980867 a) is as follows:

primers were designed and synthesized for constructing the pEC-H10-mCherry vector using the strongest Promoter H10(GCTCAACCCTTACCGGTCGGCTCTAAGCCGGCGGCGTATGGTAAGCTCTGTTATGTATA GTCCGAGCACGGCGAAAGGATACTC) in C.glutamicum as a template, reported by Wei et al (human library-based module combination (PLMC) technology for optimizing and hybridizing the synthesis of Corynebacterium glutamicum Applied Microbiology and Biotechnology 2018(102) 4117-4130), and the gene sequence of the mCherry protein and the sequence of the expression vector pEC-XK99E in C.glutamicum. Primer design was as follows (synthesized by guangzhou jinweizhi corporation):

primer 1: 5'-ggatctagagtcgacctgcag-3', respectively;

primer 2: 5'-ttaACTAGTattgcgttgcgctcac-3', respectively;

primer 3: 5'-caatACTAGTtaatgtgagttagcgcg-3';

primer 4: 5'-AGAGCTTACCATACGCCGCCGGCTTAGAGCCGACCGGTAAGGGTTGAGCctagaggatccccgggtac-3', respectively;

primer 5: 5'-GCGTATGGTAAGCTCTGTTATGTTATGTATAGTCCGAGCACGGCGAAAGGATACTCatgcgtaaaggagaagaag-3', respectively;

primer 6: 5'-cgactctagatccgccaaaacagcc-3' is added.

The construction method comprises the following steps: using pEC-XK99E as a template, and using a primer 1 and a primer 2 to amplify a skeleton region (6743bp) of the pEC plasmid; amplifying a fragment (387bp) containing an upstream region of the H10 promoter by using a promoter H10 as a template and using a primer 3 and a primer 4; a fragment (810bp) containing the region downstream of the H10 promoter and the mCherry gene was amplified using primer 5 and primer 6, using plasmid pBblactam (Zhang et al, Development of a transcription factor based lactic biosensor. ACS synthetic biology 2017, 6, 439 445) containing the mCherry gene as a template.

After PCR amplification, bands with the lengths of 387bp, 810bp and 6743bp are respectively obtained, agarose gel electrophoresis is respectively carried out on the three bands for gel cutting and recovery, and after recovery, fusion PCR amplification is carried out by using DNA fragments of the bands with the lengths of 387bp and 810bp as templates and using a primer 3 and a primer 6. And (3) cutting and recovering the obtained PCR amplified fragment. Then, the recovered fragment (i.e., the fragment containing H10 and mCherry) and the pEC framework fragment are subjected to enzyme digestion (SpeI, XbaI), and the fragment is purified by passing through a column after the enzyme digestion is finished. The method comprises the following steps of 1: 2, mixing the pEC skeleton fragment with the fragment containing H10 and mCherry, adding T4 DNA ligase reaction liquid, connecting for 1H at 16 ℃, transferring the fragment into an Escherichia coli DH5 alpha strain, and obtaining a recombinant vector with a correct sequence, namely the pEC-H10-mCherry vector.

pk18 vector: addgene, Inc., having a product number of MLCC 1103.

Corynebacterium glutamicum (C.glutamicum) in the examples belowCorynebacterium glutamicum) CGMCC No.12856, with the strain number YP097158, has been deposited in China general microbiological culture Collection center (CGMCC) at 2016, 8, 16 and the preservation number CGMCC No. 12856.

Corynebacterium glutamicum (C. glutamicum) in the examples belowCorynebacterium glutamicum) CGMCC No.21220 with strain number YPGLU001, which has been preserved in China general microbiological culture Collection center (CGMCC) at 11/23 of 2020 with a preservation number of CGMCC No. 21220.

Corynebacterium glutamicum (C.glutamicum) in the examples belowCorynebacterium glutamicum) CGMCC No.21260, with a strain number of YPFV1, which has been preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) in 11 months and 30 days of 2020, with a preservation number of CGMCC No. 21260.

Example 1, EP6 have promoter activity.

Firstly, constructing a pEC-PyfjB-mCherry vector for promoting mCherry expression by a strong promoter PyfjB in escherichia coli.

pEC-H10-mCherry vector is used as a skeleton, NEBuilder recombination technology is adopted to construct pEC-PyfjB-mCherry vector containing Escherichia coli W3110 yfjB promoter (SEQ ID No. 1: gaatttctccgcgtttttttcgcattcatctcgctaacttcgcttattatggggatcagtttcagggtttcaagggaagcactcacattgtcatcaatcttcgcaacaaggacctcggaaaa), PyfjB promoter strength is detected according to the expression condition of report protein mCherry, and PyfjB promoter strength is used as the control treatment of the sequence of the subsequent mutant promoter. The primer sequences were as follows (synthesized by Shanghai Invitrogen corporation):

pEC01：5′-ATGCGTAAAGGAGAAGAAGATAAC-3′；

pEC02：5′-CTAGAGGATCCCCGGGTAC-3′；

P1：5′-CATGGAATTCGAGCTCGGTACCCGGGGATCCTCTAGGAATTTCTCCGCGTTTTTTT-3′；

P2：5′-AATGATAGCCATGTTATCTTCTTCTCCTTTACGCATTTTTCCGAGGTCCTTGTTGC-3′。

the construction method comprises the following steps: pEC-H10-mCherry vector framework region 7772bp is obtained by amplification of primers pEC01 and pEC02 by taking pEC-H10-mCherry vector as a template; a yfjB promoter (PyfjB for short) fragment 194bp containing a pEC-H10-mCherry vector homology arm is obtained by amplification by using a DNA fragment shown in SEQ ID No.1 as a template and primers P1 and P2.

And (3) separating and purifying the two DNA fragments (the framework region of the pEC-H10-mChery vector and the PyfjB fragment containing the homology arm of the pEC-H10-mChery vector) by agarose gel electrophoresis, connecting the two DNA fragments for 30 min at 50 ℃ by NEBuilder enzyme (NEB company), identifying a single clone grown after a connecting product transforms Escherichia coli DH5a by a primer pECF/R (pECF: 5'-GTACCCGGGGATCCTCTAG-3', pECR: 5'-GTTATCTTCTTCTCCTTTACGCAT-3'), and extracting a plasmid to obtain a positive recombinant vector with a correct sequence, namely pEC-PyfjB-mChery.

The sequence of the recombinant vector pEC-PyfjB-mCherry is SEQ ID No.2 in a sequence table, wherein the yfjB promoter starts the expression of a reporter gene mCherry, and the fluorescence value of the mCherry is detected to obtain the strength of the yfjB promoter.

Secondly, constructing a mutant yfjB promoter expression vector pEC-EP 6-mCherry.

The PyfjB mutation shown in SEQ ID No.1 is used to obtain the EP6 promoter with the size of 124bp, and the sequence of the EP6 promoter is SEQ ID No. 3.

The vector construction was carried out by NEBuilder recombination technique, and the primers were designed as follows (synthesized by Shanghai Invitrogen corporation):

P5：5′-CATGGAATTCGAGCTCGGTACCCGGGGATCCTCTAGGAATTTCTTCGCGTTTTTTT-3′；

P6：5′-AATGATAGCCATGTTATCTTCTTCTCCTTTACGCATGATTTGGAGGTCCCTGCGTT-3′。

the construction method comprises the following steps: using pEC-PyfjB-mCherry vector as a template, and amplifying 7772bp of a framework region of pEC-H10-mCherry vector by using primers pEC01 and pEC 02; the DNA fragment shown in SEQ ID No.3 is used as a template, and primers P5 and P6 are used for amplifying an EP6 promoter fragment 196bp containing a pEC-H10-mCherry vector homology arm.

The two DNA fragments (the framework region of the pEC-H10-mCherry vector and the EP6 promoter fragment containing the homology arm of the pEC-H10-mCherry vector) are separated and purified by agarose gel electrophoresis, then are connected for 30 min at 50 ℃ by NEBuilder enzyme (NEB company), a single clone grown after the connection product is transformed into Escherichia coli DH5a is identified by a primer pECF/R (pECF: 5'-GTACCCGGGGATCCTCTAG-3', pECR: 5'-GTTATCTTCTTCTCCTTTACGCAT-3'), and plasmids are extracted to obtain a positive recombinant vector with correct sequence, which is marked as pEC-EP 6-mChery.

pEC-EP6-mCherry is a recombinant vector obtained by replacing PyfjB (SEQ ID No. 1) of pEC-PyfjB-mCherry with EP6 (SEQ ID No. 3).

Thirdly, measuring the activity of the promoter.

Respectively introducing the pEC-EP6-mCherry and the pEC-PyfjB-mCherry vector in the step two into wild Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC13032 to obtain recombinant bacteria ATCC13032/pEC-EP6-mCherry and ATCC 13032/pEC-PyfjB-mCherry; respectively introducing the pEC-EP6-mCherry vector in the step two and the pEC-PyfjB-mCherry vector in the step one into wild Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC13869 to obtain recombinant bacteria ATCC13869/pEC-EP6-mCherry and ATCC 13869/pEC-PyfjB-mCherry; and respectively introducing the pEC-EP6-mCherry and the pEC-PyfjB-mCherry vector in the step two into wild Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC14067 to obtain recombinant bacteria ATCC14067/pEC-EP6-mCherry and ATCC 14067/pEC-PyfjB-mCherry. The strength of the two promoters in C.glutamicum was compared.

And respectively inoculating the monoclonals of the 6 recombinant bacteria into a 96-deep-well plate containing 900 mu L LBHIS culture medium in an equal amount, wherein each monoclonals contains 3 repeats, culturing at 30 ℃ and 800rpm for 24h, detecting the fluorescence value of mCherry by using a microplate reader, and comparing the promoter activities of yfjB and EP6 according to the fluorescence intensity.

LBHIS Medium: 5g Tryptone/L, 5g NaCl/L, 2.5g yeast extract/L, 18.5g BHI (Brain Heart Infusion)/L, 91g sorbitol/L, pH 7.2.

The results show that the EP6 promoter has promoter activity, and its promoter activity is significantly higher than that of the yfjB promoter (FIG. 1).

Example 2 replacement of the lysA gene promoter of the lysine metabolic pathway in Corynebacterium glutamicum and its effect on lysine production.

Firstly, preparing recombinant bacteria.

The promoter EP6 and the promoter yfjB are respectively replaced by corynebacterium glutamicum (C) with high lysine yield by a homologous recombination method by adopting a pk18 vectorCorynebacterium glutamicum) The original promoter (1-148 th position of SEQ ID No. 4) of lysA which is a key gene of a lysine metabolism pathway in CGMCC No.12856 is utilized to start expression of lysA in the lysine metabolism pathway by utilizing an exogenous promoter, so that lysine-producing bacteria with exogenous promoter-gene combination are obtained, and fermentation tests of the producing bacteria show that a strain starting lysA by EP6 can increase the yield of lysine higher, and the strain is named as YPL-4-042. The sequence of lysA at position 149-1486 of SEQ ID No. 4.

The vector is constructed by NEBuilder recombinant technology, and the primers are designed as follows (synthesized by Shanghai invitrogen company):

P7：5′-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGCCCAGGTGAAGAAGTCGTTG-3′；

P8：5′-AAAAAAACGCGGAGAAATTCATGCCCCGTTCGACAATAAA-3′；

P9：5′-TTTATTGTCGAACGGGGCATGAATTTCTCCGCGTTTTTTT-3′；

P10：5′-GCGAGATCAGCTGGTGTCATTTTTCCGAGGTCCTTGTTGC-3′；

P11：5′-GCAACAAGGACCTCGGAAAAATGACACCAGCTGATCTCGC-3′；

P12：5′-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCTCGAACATCATCTCCACGCC-3′；

P8E：5′-AAAAAAACGCGAAGAAATTCATGCCCCGTTCGACAATAAA-3′；

P9E：5′-TTTATTGTCGAACGGGGCATGAATTTCTTCGCGTTTTTTT-3′；

P10E：5′-GCGAGATCAGCTGGTGTCATGATTTGGAGGTCCCTGCGTT-3′；

P11E：5′-AACGCAGGGACCTCCAAATCATGACACCAGCTGATCTCGC-3′。

the specific method of homologous recombination is as follows:

carrying out PCR amplification by using Corynebacterium glutamicum ATCC13032 as a template and using a primer P7/P8 (the sequence of a PCR product is SEQ ID No.5, 1 st to 801 th sites) to obtain an upstream homologous arm fragment 801 bp; taking the vector pEC-PyfjB-mCherry as a template, and carrying out PCR amplification by using a primer P9/P10 to obtain PyfjB 162bp (the sequence of a PCR product is 762-923 th site of SEQ ID No. 5); the downstream homology arm fragment 765bp was obtained by PCR amplification using Corynebacterium glutamicum ATCC13032 as template and primer P11/P12 (the PCR product sequence is at 884-1648 of SEQ ID No. 5).

After the PCR reaction is finished, the obtained 3 PCR products are respectively subjected to electrophoresis recovery by adopting a column type DNA gel recovery kit. The 3 recovered PCR products are subjected toXbalI andBamHi after digestion, purified pK18mobsacB vector (Addgene, the vector contains kanamycin resistance as a screening marker) is connected for 30 min at 50 ℃ by NEBuilder Enzyme (NEB), a single clone grown after the connection product is transformed into Escherichia coli DH5a is identified by PCR by using M13 primer (M13F: 5'-TGTAAAACGACGGCCAGT-3', M13R: 5'-CAGGAAACAGCTATGACC-3') to obtain a positive integration vector (recombinant vector), the recombinant vector with correct sequence is extracted and is marked as pK 18-PyfjB-lysAEE, the recombinant vector contains a kanamycin resistance marker, and a recombinant vector integrated into a genome can be obtained by kanamycin screening.

pK 18-PyfjB-lysAEE contains the DNA fragment shown in SEQ ID No.5, wherein in SEQ ID No.5, the 1 st-801 th position is the sequence of the upstream homology arm fragment, the 884 nd-1648 th position is the sequence of the downstream homology arm fragment, and the 762 nd-923 th position is the sequence of PyfjB.

Carrying out PCR amplification by using Corynebacterium glutamicum ATCC13032 as a template and using a primer P7/P8E (1 st-801 th position of SEQ ID No. 6) to obtain an upstream homology arm fragment 801 bp; PCR amplification is carried out by taking pEC-EP6-mCherry as a template and P9E/P10E to obtain EP 6164 bp (SEQ ID No.6, 762) -925 bit); PCR amplification was performed using Corynebacterium glutamicum ATCC13032 as template and primer P11E/P12 (position 886-1650 of SEQ ID No. 6) to obtain 765bp of downstream homology arm fragment.

After the PCR reaction is finished, the obtained 3 PCR products are respectively subjected to electrophoresis recovery by adopting a column type DNA gel recovery kit. The 3 recovered PCR products are subjected toXbalI andBamHi after digestion, purified pK18mobsacB vector (Addgene, the vector contains kanamycin resistance as a selection marker) is connected for 30 min at 50 ℃ by NEBuilder Enzyme (NEB), a single clone grown after the connection product is transformed into Escherichia coli DH5a is identified by PCR by using M13 primer (M13F: 5'-TGTAAAACGACGGCCAGT-3', M13R: 5'-CAGGAAACAGCTATGACC-3') to obtain a positive integration vector (recombinant vector), the recombinant vector with correct sequence is extracted and is marked as pK18-EP 6-lysAEE, the recombinant vector contains kanamycin resistance marker, and a recombinant vector integrated into a genome can be obtained by kanamycin screening.

pK18-EP 6-lysAEE comprises the DNA fragment shown in SEQ ID No.6, wherein in the SEQ ID No.6, the 1 st-801 th position is the sequence of the upstream homology arm fragment, the 886 nd-1650 th position is the sequence of the downstream homology arm fragment, and the 762 nd-925 th position is the sequence of EP 6.

Respectively and electrically transforming recombinant vectors pK 18-PyfjB-lysAEE and pK18-EP 6-lysAEE with correct sequencing into Corynebacterium glutamicum CGMCC No.12856 with high lysine yield, culturing in a recovery culture medium according to recovery culture conditions, carrying out PCR identification on single colonies generated by culture by using a P13/P14 primer, and carrying out PCR amplification on recombinant bacteria introduced with pK 18-PyfjB-lysAEE to obtain a positive strain with an 885bp (SEQ ID No. 7) fragment, wherein the positive strain is marked as CGMCC No.12856/pK 18-PyfjB-lysAEE; the recombinant bacteria introduced with pK18-EP 6-lysAEE are PCR amplified to obtain a positive strain with a 885bp (SEQ ID No. 8) fragment, which is marked as CGMCC No.12856/pK18-EP 6-lysAEE, and the original strain with no fragment amplified is obtained.

Culturing the positive strain on a culture medium containing 15% of sucrose, carrying out PCR identification on the single colony generated by the culture by adopting a P15/P16 primer, amplifying a strain with the size of 841bp (SEQ ID No. 9) which is a positive strain of a Corynebacterium glutamicum CGMCC No.12856lysA original promoter with high-yield lysine replaced by PyfjB, and an amplified strain with the size of 843bp (SEQ ID No. 10) which is a positive strain of a Corynebacterium glutamicum CGMCC No.12856lysA original promoter with high-yield lysine replaced by EP6, wherein the strain without fragment amplification is a protobacteria.

Positive strains obtained from recombinant bacteria introduced with pK 18-PyfjB-lysAEE and pK18-EP 6-lysAEE were designated YPL-4-041 (PyfjB-lysA integrated strain) and YPL-4-042 (EP 6-lysA integrated strain), respectively.

YPL-4-041 (PyfjB-lysA integrated strain) is a recombinant strain obtained by replacing the original promoter of lysA (position 1-148 of SEQ ID No. 4) of Corynebacterium glutamicum CGMCC No.12856 of a high-yield lysine with yfjB promoter and keeping other nucleotides in the genome unchanged, and can improve the yield of lysine; YPL-4-042 (EP 6-lysA integration strain) is a recombinant strain obtained by replacing a Corynebacterium glutamicum CGMCC No.12856lysA original promoter (positions 1-148 of SEQ ID No. 4) which produces lysine with EP6 promoter and keeping other nucleotides in the genome unchanged, and EP6 promoter is used as a mutant yfjB promoter which promotes expression of lysA and can further improve the yield of lysine.

The PCR identification primers are shown below:

p13: 5'-GGCGACGATCTTGTTTGACC-3' (inside the cg1332 gene);

p14: 5'-ACTGATCCCCATAATAAGCG-3' (PyfjB internal);

p15: 5'-CGCTTATTATGGGGATCAGT-3' (PyfjB internal);

p16: 5'-GTAGTAGACATCGAAATCGG-3' (inside the argS gene).

And (3) recovering the culture medium: the solvent is water, and the solute and the content thereof in the recovery culture medium are respectively 5g/L glucose, 3g/L urea, 10g/L yeast powder, 10 mu g/L biotin, 15g/L soybean meal, 0.5g/L succinic acid, 1g/L monopotassium phosphate, 2.5g/L sodium chloride, 91g/L sorbitol, 18.5g/L brain and heart infusion and pH7.0.

Medium with 15% sucrose: the solvent is water, the solute and the content of the solute in a 15% sucrose culture medium are respectively 15g/L sucrose, 3g/L urea, 10g/L yeast powder, 10 mu g/L biotin, 15g/L soybean meal, 0.5g/L succinic acid, 1g/L potassium dihydrogen phosphate, 2.5g/L sodium chloride, 91g/L sorbitol, 18.5g/L brain-heart infusion, 15g/L agar powder and pH7.0.

Recovering culture conditions: resuspending the cells in a recovery medium at 46 ℃, transferring to an EP tube, shaking at 220rpm and 37 ℃ for 45-60min, and then transferring to a shaking table at 30 ℃ for 45-60min with constant rotating speed.

And II, performing an L-lysine fermentation experiment.

The strains YPL-4-041, YPL-4-042 and the original strain Corynebacterium glutamicum CGMCC No.12856 were subjected to fermentation experiments in a BLBIO-5GC-4-H type fermenter (Bailan Biotech Co., Ltd., Shanghai) with a fermentation medium 1 and culture conditions 1, and L-lysine production was measured after the fermentation was completed, and each strain was repeated three times.

The yield of L-lysine was determined by high performance liquid chromatography.

Fermentation medium 1: the solvent is water, the solute and the content of the solute in the fermentation medium 1 are respectively 30g/L of starch hydrolysis sugar, 12g/L of ammonium sulfate, 0.87g/L of magnesium sulfate, 20g/L of molasses, 3mL/L of acidified corn steep liquor, 0.4mL/L of phosphoric acid, 0.53g/L of potassium chloride, 4mL/L of antifoaming agent (2% of foam), 120mg/L of ferrous sulfate, 120mg/L of manganese sulfate, 42mg/L of nicotinamide, 6.3mg/L of calcium pantothenate, 16.3 mg/L of vitamin B, 0.6g/L of copper sulfate, 0.6g/L of zinc sulfate and 0.88mg/L of biotin. The acidified corn steep liquor is a product of Ningxia Yipin Biotechnology GmbH.

Culture conditions 1:

correcting DO to be 100%, calibrating after the temperature is 37 ℃, the air volume is 4L/min, the rotating speed is 1000rpm, the tank pressure is 0Mpa and the time is 5 min;

inoculation amount: 10 percent;

the culture temperature is as follows: 37 ℃;

pH：6.9±0.05；

dissolved oxygen DO: 10 to 30 percent;

initial conditions: the temperature is 37 ℃, the pH value is 6.9, the tank pressure is 0Mpa, the air volume is 3L/min, and the rotating speed is 550 rpm;

and (3) whole-process control: when the whole process is controlled to be 1 and the dissolved oxygen is less than 30 percent, the rotating speed is increased to 750rpm → 800rpm → the air volume is increased to 4L/min → 850rpm → 950rpm in sequence; 2. fermenting for 6h, and extracting the tank pressure to 0.01 Mpa; the pressure for extracting the tank is 0.02Mpa → 0.03Mpa → 0.04Mpa → 0.05Mpa for 12 h;

residual sugar control: 0.1-0.2% of F12 h; controlling residual sugar to be 0.1-0.05% after F12h by combining DO requirement;

ammonia nitrogen control: 0.1-0.15 of F12 h; F12-F32h0.15-0.25; 0.1-0.15 after F32 h;

feeding materials in a flowing manner: 25% of ammonia water, 70% of concentrated sugar, 50% of ammonium sulfate and 10% of foam enemy;

and (3) fermentation period: about 48 h.

The result shows that the L-lysine yield of the strain CGMCC No.12856 is 18.9g/100 mL; the yield of the L-lysine of the YPL-4-041 is 19.2g/100mL, which is obviously higher than that of a strain CGMCC No.12856 (P < 0.05); the L-lysine yield of YPL-4-042 is 19.7g/100mL, which is obviously higher than that of the strain CGMCC No.12856 (P < 0.05). Thus, it was shown that the replacement of lysA original promoter with yfjB or EP6 promoter in lysine-producing bacterium CGMCC No.12856 can increase the yield of lysine, and that the lysine yield is higher as mutant EP6 promotes the expression of lysA.

Example 3, increase of promoter of BBD29_14295 gene of glutamic acid metabolism pathway in Corynebacterium glutamicum and its effect on glutamic acid production.

Firstly, preparing recombinant bacteria.

The promoters EP6 and yfjB were respectively substituted for Corynebacterium glutamicum (C.glutamicum) of high glutamic acid yield by homologous recombination using pk18 vectorCorynebacterium glutamicum) The original promoter (1-36 site of SEQ ID No. 11) of key gene BBD29_14295 of glutamic acid metabolism pathway in CGMCC No.21220 is utilized to start the expression of BBD29_14295 in glutamic acid metabolism pathway to obtain glutamic acid producing bacteria of exogenous promoter-gene combination, and the fermentation test of the producing bacteria finds that the bacterial strain starting BBD29_14295 by EP6 can increase the yield of glutamic acid higher, and the bacterial strain is named as YPG-092. 37-116 of SEQ ID No.11Bit 1 is the sequence of BBD29_ 14295.

The vector was constructed by NEBuilder recombinant technology, and the primers were designed as follows (synthesized by Shanghai Invitrogen corporation):

P17：5′-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGCGGAAACAACGCAGCCATAG-3′；

P18：5′-AAAAAAACGCGGAGAAATTCTTAGAGGAATTTAACGCCTT-3′；

P19：5′-AAGGCGTTAAATTCCTCTAAGAATTTCTCCGCGTTTTTTT-3′；

P20：5′-TCGTCGATTAGCTCGAACATTTTTCCGAGGTCCTTGTTGC-3′；

P21：5′-GCAACAAGGACCTCGGAAAAATGTTCGAGCTAATCGACGA-3′；

P22：5′-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCATGACGTCCTGGAGCGCTGC-3′；

P18E：5′-AAAAAAACGCGAAGAAATTCTTAGAGGAATTTAACGCCTT-3′；

P19E：5′-AAGGCGTTAAATTCCTCTAAGAATTTCTTCGCGTTTTTTT-3′；

P20E：5′-TCGTCGATTAGCTCGAACATGATTTGGAGGTCCCTGCGTT-3′；

P21E：5′-AACGCAGGGACCTCCAAATCATGTTCGAGCTAATCGACGA-3′。

the specific method of homologous recombination is as follows:

carrying out PCR amplification by using Corynebacterium glutamicum (ATCC 13869) as a template and a primer P17/P18 (SEQ ID No.12, 1-792) to obtain an upstream homologous arm fragment 792 bp; carrying out PCR amplification by using the vector pEC-PyfjB-mCherry as a template and a primer P19/P20 to obtain PyfjB 162bp (SEQ ID No.12, 753-914 site); the downstream homology arm fragment 825bp was obtained by PCR amplification using Corynebacterium glutamicum ATCC13869 as template and primer P21/P22 (SEQ ID No.12 at position 875-1699).

After the PCR reaction is finished, the obtained 3 PCR products are respectively subjected to electrophoresis recovery by adopting a column type DNA gel recovery kit. 3 kinds of recovered PCR products and their preparationXbalI andBamHthe pK18mobsacB vector (Addgene, containing kanamycin resistance as a selection marker) purified after I cleavage was purified using NEBuilder Enzyme (NE)Company B) was ligated at 50 ℃ for 30 min, and the ligation product was transformed into e.coli DH5a, and the single clone was grown using M13 primer (M13F: 5'-TGTAAAACGACGGCCAGT-3', M13R: 5'-CAGGAAACAGCTATGACC-3') and identifying by PCR to obtain a positive integration vector (recombinant vector), marking the obtained recombinant vector with correct sequence as pK18-PyfjB-BBD 29-14295 OE, wherein the recombinant vector contains a kanamycin resistance marker, and a recombinant of the vector integrated on a genome can be obtained by kanamycin screening.

pK18-PyfjB-BBD29_14295OE comprises the DNA fragment shown in SEQ ID No.12, wherein the 1 st-792 st position in the SEQ ID No.12 is the sequence of the upstream homology arm fragment, the 875 nd-1699 th position in the SEQ ID No.12 is the sequence of the downstream homology arm fragment, and the 753 nd-914 nd position in the sequence of PyfjB.

Carrying out PCR amplification by using Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC13869 as a template and using a primer P17/P18E (SEQ ID No.13, 1-792) to obtain an upstream homologous arm fragment 792 bp; using the vector pEC-EP6-mCherry as a template and primer P19E/P20E for PCR amplification to obtain EP 6164 bp (position 753-916 of SEQ ID No. 13); the downstream homology arm fragment 825bp was obtained by PCR amplification using Corynebacterium glutamicum ATCC13869 as template and primer P21E/P22 (SEQ ID No.13, position 877-1701).

After the PCR reaction is finished, the obtained 3 PCR products are respectively subjected to electrophoresis recovery by adopting a column type DNA gel recovery kit. 3 kinds of recovered PCR products and their preparationXbalI andBamHi after enzyme digestion, purified pK18mobsacB vector (Addgene company, the vector contains kanamycin resistance as a screening marker) is connected for 30 min at 50 ℃ by NEBuilder enzyme (NEB company), a positive integration vector (recombinant vector) is obtained by PCR identification of a single clone grown after the connection product is transformed into Escherichia coli DH5a by using M13 primer (M13F: 5'-TGTAAAACGACGGCCAGT-3', M13R: 5'-CAGGAAACAGCTATGACC-3'), the obtained recombinant vector with correct sequence is marked as pK18-EP6-BBD29_14295OE, the recombinant vector contains the kanamycin resistance marker, and a recombinant vector integrated on a genome can be obtained by kanamycin screening.

pK18-EP6-BBD29_14295OE contains the DNA fragment shown in SEQ ID No.13, wherein the 1 st to 792 st positions in the SEQ ID No.13 are the sequence of the upstream homology arm fragment, the 877 nd to 1701 th positions are the sequence of the downstream homology arm fragment, and the 753 nd to 916 th positions are the sequence of EP 6.

The recombinant vectors (pK 18-PyfjB-BBD29_14295OE and pK18-EP6-BBD29_14295 OE) with correct sequencing are respectively and electrically transformed into Corynebacterium glutamicum CGMCC No.21220 with high glutamic acid yield, the Corynebacterium glutamicum CGMCC No.21220 is cultured in a recovery culture medium (same as example 2) according to recovery culture conditions (same as example 2), a single colony generated by culture is identified by PCR through P23/P24 primers, the PyfjB-BBD29_14295 integrating bacterium is used as a positive strain of 849bp (SEQ ID No. 14) fragment amplified by PCR, the EP6-BBD29_14290 integrating bacterium is used as a positive strain of 849bp (SEQ ID No. 15) fragment amplified by PCR, and the original bacterium is used as the original bacterium without fragment amplified.

Culturing the positive strain on a culture medium containing 15% of sucrose (the same as the culture medium containing 15% of sucrose in example 2), and performing PCR identification on the single colony generated by culture by further adopting a P25/P26 primer to amplify a strain with the size of 889bp (SEQ ID No. 16) which is a strain with PyfjB replacing a BBD 29-14295 original promoter on a Corynebacterium glutamicum CGMCC number 21220 genome of high-yield glutamic acid; the strain with the amplified size of 891bp (SEQ ID No. 17) is a strain of a BBD29_14295 original promoter on a Corynebacterium glutamicum CGMCC No.21220 genome with high-yield glutamic acid replaced by EP6, and the strain without amplified fragment is an original strain.

A recombinant strain in which the BBD29_14295 original promoter was replaced by PyfjB was designated YPG-091 (PyfjB-BBD 29_14295 integration strain), and a recombinant strain in which the BBD29_14295 original promoter was replaced by EP6 was designated YPG-092 (EP 6-BBD29_14295 integration strain).

YPG-091 (PyfjB-BBD 29_14295 integration strain) is a recombinant strain obtained by replacing the original promoter (1-36 th position of SEQ ID No. 11) of Corynebacterium glutamicum CGMCC No.21220 BBD29_14295 with PyfjB for high-yield glutamic acid, and keeping other nucleotides in the genome unchanged, and can improve the yield of glutamic acid; YPG-092 (EP 6-BBD29_14295 integration strain) is a recombinant strain obtained by replacing the original promoter (1-36 th position of SEQ ID No. 11) of Corynebacterium glutamicum CGMCC No.21220 BBD29_14295 with EP6, which can produce high-yield glutamic acid, and keeping other nucleotides in the genome unchanged, and EP6 promoter is used as mutant yfjB promoter, which can promote expression of BBD29_14295 to improve the yield of glutamic acid.

The PCR identification primers are shown below:

p23: 5'-GAAGTAGGTC CTCGTGTTGC-3' (inside the BBD29_14290 gene);

p24: 5'-ACTGATCCCC ATAATAAGCG-3' (PyfjB internal);

p25: 5'-CGCTTATTAT GGGGATCAGT-3' (PyfjB internal);

p26: 5'-GACGAGTTGT GCTTGTAGTC-3' (inside the BBD29_14295 gene).

And secondly, performing an L-glutamic acid fermentation experiment.

The strains YPG-091, YPG-092 and the original strain CGMCC No.21220 are subjected to fermentation experiments in a BLBIO-5GC-4-H type fermentation tank (Shanghai Bailun Biotech Co., Ltd.) by using a fermentation medium 2 and a culture condition 2, and the yield and OD value of L-glutamic acid are detected after the fermentation is finished. Each strain was replicated three times.

The glutamic acid production was measured by a biosensor.

Fermentation medium 2: the solvent is water, the solute and its concentration in the fermentation medium 2 are glucose 5.0g/L, phosphoric acid 0.38g/L, magnesium sulfate 1.85g/L, potassium chloride 1.6g/L, biotin 550 μ g/L, vitamin B1300 μ g/L, ferrous sulfate 10mg/L, manganese sulfate 10g/dl, KH ₂ PO ₄ 2.8g/L, 0.75mg/L of vitamin C, 122.5 mu g/L of vitamin B, 0.75mg/L of p-aminobenzoic acid, 0.0015mL/dL of antifoaming agent, 1.5g/L of betaine, 7mL/L of cane molasses, 77mL/L of corn steep liquor and 1.7g/L of aspartic acid.

Culture conditions 2:

correcting DO to be 100%, calibrating after the temperature is 32.5 ℃, the air volume is 7L/min, the rotating speed is 700rpm and the tank pressure is 0Mpa for 5 min;

inoculation amount: 13 percent; 0 h: the rotating speed is 400rpm, the air volume is 3L/min, the pressure is 0.05MPA, and the culture temperature is 32.5 ℃;

bacterial liquid concentration OD = 1.0: the rotating speed is 600rpm, the air volume is 5L/min, the pressure is 0.08MPA, and the culture temperature is 37 ℃;

bacterial liquid concentration OD = 1.0-bacterial liquid concentration OD = 1.4: the rotating speed is 700rpm, the air volume is 7L/min, the pressure is 0.11MPA, and the culture temperature is 38 ℃;

after the fermentation is finished within 32-34 h, controlling the process to take dissolved oxygen of 50-20% as the standard for improving and reducing the air volume;

pH: controlling 7.0 for 0h and 6.8 for 14 h;

sugar feeding control: the concentration of sugar in the fermentation tank is 50-55%, and the residual sugar in the fermentation tank is controlled to be 0.5-1.0%.

The results showed that the L-glutamic acid yield of CGMCC No.21220 was 102.1g/L and the OD (562nm) was 46.1; the yield of the L-glutamic acid of YPG-091 is 103.3g/L, which is obviously higher than that of a strain CGMCC No.21220 (P is less than 0.05), and the OD (562nm) is 46.6; the yield of L-glutamic acid of YPG-092 is 105.1g/L, which is obviously higher than that of strain CGMCC No.21220 (P < 0.05), and OD (562nm) is 47.1. Thus, it was shown that the replacement of BBD29_14295 original promoter with yfjB or EP6 promoter in glutamic acid-producing bacterium CGMCC No.21220 can increase the glutamic acid yield, and that as mutated EP6 promotes the expression of BBD29_14295, the glutamic acid yield is higher.

Example 4 increase of the promoter of the ilvC gene of the valine metabolic pathway in Corynebacterium glutamicum and its effect on valine production.

Firstly, preparing recombinant bacteria.

The promoter EP6 and yfjB are respectively replaced by Corynebacterium glutamicum (C.glutamicum) which can produce valine with high yield by adopting a pk18 vector through a homologous recombination methodCorynebacterium glutamicum) The original promoter (1-179 site of SEQ ID No. 18) of ilvC, which is a key gene of a valine metabolic pathway in CGMCC No.21260, is utilized to promote the expression of ilvC gene in a glutamic acid metabolic pathway by utilizing an exogenous promoter so as to obtain valine-producing bacteria with exogenous promoter-gene combination, and fermentation tests of the production bacteria show that the strain with EP6 promoting ilvC can improve the yield of valine more, and the strain is named as YPV-098. The sequence ilvC at position 180-1196 of SEQ ID No. 18.

P27：5′-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGAGAGATCAACGACCGCCCAG-3′；

P28：5′-AAAAAAACGCGGAGAAATTCTTAGATCTTGGCCGGAGCCA-3′；

P29：5′-TGGCTCCGGCCAAGATCTAAGAATTTCTCCGCGTTTTTTT-3′；

P30：5′-TAAAGCAGTTCAATAGCCATTTTTCCGAGGTCCTTGTTGC-3′；

P31：5′-GCAACAAGGACCTCGGAAAAATGGCTATTGAACTGCTTTA-3′；

P32：5′-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCTGAGGCGCTTGGTGAAGGTG-3′；

P28E：5′-AAAAAAACGCGAAGAAATTC TTAGATCTTGGCCGGAGCCA -3′；

P29E：5′-TGGCTCCGGCCAAGATCTAAGAATTTCTTCGCGTTTTTTT-3′；

P30E：5′-TAAAGCAGTTCAATAGCCATGATTTGGAGGTCCCTGCGTT-3′；

P31E：5′-AACGCAGGGACCTCCAAATCATGGCTATTGAACTGCTTTA-3′。

the specific method of homologous recombination is as follows:

carrying out PCR amplification by using Corynebacterium glutamicum ATCC14067 as a template and using a primer P27/P28 (SEQ ID No.19, 1 st to 805 th sites) to obtain an upstream homology arm fragment 805 bp; the vector pEC-PyfjB-mCherry is taken as a template, the primer P29/P30 is used for PCR amplification to obtain PyfjB 162bp (position 766 and 927 of SEQ ID No. 19) and the primer P31/P32 (position 888 and 1822 of SEQ ID No. 19) which are taken as templates and are used for PCR amplification to obtain the downstream homologous arm fragment 935 bp.

After the PCR reaction is finished, the obtained 3 PCR products are respectively subjected to electrophoresis recovery by adopting a column type DNA gel recovery kit. 3 kinds of recovered PCR products and their synthesis processXbalI andBamHi enzyme digestion of purified pK18mobsacB vector (Addgene company, the vector contains kanamycin resistance as a screening marker) and NEBuilder enzyme (NEB company) at 50 ℃ for 30 min, the ligation product is transformed into Escherichia coli DH5a, the grown single clone is identified by PCR using M13 primer (M13F: 5'-TGTAAAACGACGGCCAGT-3', M13R: 5'-CAGGAAACAGCTATGACC-3') to obtain positive integration vector (recombinant vector), the obtained recombinant vector with correct sequence is marked as pK18-PyfjB-ilvCOE, the recombinant vector contains kanamycin resistance markerKanamycin selection yielded recombinants with vector integration into the genome.

pK18-PyfjB-ilvCOE contains the DNA fragment shown in SEQ ID No.19, wherein the 1 st to 805 nd positions of the SEQ ID No.19 are the sequences of the upstream homology arm fragments, the 888 nd 1822 nd positions are the sequences of the downstream homology arm fragments, and the 766 nd position 927 nd positions are the sequences of PyfjB.

Carrying out PCR amplification by using Corynebacterium glutamicum ATCC14067 as a template and using a primer P27/P28E (SEQ ID No.20, 1 st to 805 th sites) to obtain an upstream homology arm fragment 805 bp; carrying out PCR amplification by using a vector pEC-EP6-mCherry as a template and a primer P29E/P30E to obtain EP 6164 bp (position 766-929 of SEQ ID No. 20); the downstream homology arm fragment 935bp was obtained by PCR amplification using Corynebacterium glutamicum ATCC14067 as template and primer P31E/P32 (SEQ ID No.20, 890-1824).

After the PCR reaction is finished, the obtained 3 PCR products are respectively subjected to electrophoresis recovery by adopting a column type DNA gel recovery kit. 3 kinds of recovered PCR products and their preparationXbalI andBamHi after digestion, purified pK18mobsacB vector (Addgene, the vector contains kanamycin resistance as a selection marker) is connected for 30 min at 50 ℃ by NEBuilder Enzyme (NEB), a positive integration vector (recombinant vector) is obtained by PCR identification of a single clone grown after the connection product is transformed into Escherichia coli DH5a by using M13 primer (M13F: 5'-TGTAAAACGACGGCCAGT-3', M13R: 5'-CAGGAAACAGCTATGACC-3'), the obtained recombinant vector with correct sequence is marked as pK18-EP6-ilvCOE, the recombinant vector contains a kanamycin resistance marker, and a recombinant plasmid integrated on a genome of the vector can be obtained by kanamycin screening.

pK18-EP6-ilvCOE contains the DNA fragment shown in SEQ ID No.20, in the SEQ ID No.20, the 1 st to 805 th positions are the sequence of the upstream homology arm fragment, the 890 nd and 1824 th positions are the sequence of the downstream homology arm fragment, and the 766 nd and 929 th positions are the sequence of EP 6.

The integrated vectors (pK 18-PyfjB-ilvCOE, pK18-EP 6-ilvCOE) with correct sequencing are respectively electrically transformed into Corynebacterium glutamicum CGMCC No.21260 with high valine yield, the integrated vectors are cultured in a recovery culture medium (same as example 2) according to recovery culture conditions (same as example 2), single colonies generated by culture are subjected to PCR identification through a P33/P34 primer, the PyfjB-ilvC integrated strain is subjected to PCR amplification to obtain a strain with a 901bp (SEQ ID No. 21) fragment as a positive strain, the EP6-ilvC integrated strain is subjected to PCR amplification to obtain a strain with a 901bp (SEQ ID No. 22) fragment as a positive strain, and a strain without the fragment as a protobacteria.

Culturing the positive strain on a culture medium containing 15% of sucrose (same as the 15% sucrose culture medium in example 2), further performing PCR identification on a single colony generated by culture by adopting a P35/P36 primer, and amplifying a strain with the size of 1023bp (SEQ ID No. 23) to be a positive strain of ilvC original promoter on a genome of Corynebacterium glutamicum CGMCC No.21260 with PyfjB replacing high-yield valine; the strain with the amplified size of 1025bp (SEQ ID No. 24) is a strain of which EP6 replaces the ilvC original promoter on the genome of Corynebacterium glutamicum CGMCC No.21260 with high valine yield, and the strain without amplified fragment is the original strain.

The recombinant strain in which the ilvC original promoter was replaced with PyfjB was designated YPV-097 (PyfjB-ilvC integration strain), and the recombinant strain in which the ilvC original promoter was replaced with EP6 was designated YPV-098 (EP 6-ilvC integration strain).

YPV-097 (PyfjB-ilvC integrated strain) is a recombinant strain obtained by replacing a Corynebacterium glutamicum CGMCC No.21260 ilvC original promoter (1-179 of SEQ ID No. 18) for high yield of valine with PyfjB and keeping other nucleotides in the genome unchanged, and can improve the yield of valine; YPV-098 (EP 6-ilvC integrated strain) is a recombinant strain obtained by replacing the original promoter (1-179 of SEQ ID No. 18) of high valine-yielding Corynebacterium glutamicum CGMCC No.21260 ilvC with EP6 and keeping other nucleotides in the genome unchanged, and the EP6 promoter is used as a mutant yfjB promoter, and can promote the expression of ilvC to further improve the yield of valine.

The PCR identification primers are shown below:

p33: 5'-CCCGACTTTG TTACCCTTTC-3' (inside the CEY17_ RS06885 gene);

p34: 5'-ACTGATCCCC ATAATAAGCG-3' (PyfjB internal);

p35: 5'-CGCTTATTAT GGGGATCAGT-3' (PyfjB internal);

p36: 5'-GGGTGGTTGT TGTAGGAAGC-3' (inside the ilvC gene).

And II, performing a valine fermentation experiment.

The strains YPV-097, YPV-098 and the original strain Corynebacterium glutamicum CGMCC No.21260 were subjected to fermentation experiments in a fermentation tank (BLBIO-5 GC-4-H) model (Shanghai Bailun Biotechnology Co., Ltd.) under a fermentation medium 3 and a culture condition 3, and the L-valine yield and OD value were measured after the fermentation was completed. Each strain was replicated three times.

The detection method of the yield of the L-valine is high performance liquid chromatography.

Fermentation medium 3: the solvent is water, and the solute and the concentration thereof are respectively 14 g/L of ammonium sulfate, 1g/L of monopotassium phosphate, 1g/L of dipotassium phosphate, 0.5g/L of magnesium sulfate, 2g/L of yeast powder, 18 mg/L of ferrous sulfate, 4.2 mg/L of manganese sulfate, 0.02 mg/L of biotin, 0.5 mL/L of vitamin B12 mg/L, antifoam (CB-442) antifoaming agent and 40 g/L of 70% glucose (substrate).

Culture conditions 3:

correcting DO by 100%, at 33 deg.C, air volume of 1L/min, rotation speed of 400rpm, and tank pressure of 0.01Mpa, and calibrating after 5 min;

the inoculation amount is 3.5 percent; the culture temperature is 33 ℃;

pH7.0 + -0.05; dissolved oxygen DO 10-20%;

initial conditions: the temperature is 33 ℃, the pH value is 7.0, the tank pressure is 0Mpa, the air volume is 0.1L/min, and the rotating speed is 400 rpm;

and (3) whole-process control: the temperature is 33 ℃, the pH value is 7.0, the tank pressure is 0Mpa, the air volume is 0.2L/min, and the rotating speed is 400 rpm;

controlling residual sugar: 0.1-0.2% of F12 h; controlling residual sugar to be less than or equal to 0.02% by combining DO requirement after F12 h;

and (4) culturing and maturing standard: OD ₆₁₀ 30-35；OD ₆₁₀ Stopping ventilation and standing for 2 hours after the time is more than 30 (according to the purpose of batch experiment, carrying out thallus separation or continuous catalysis);

feeding materials in a flowing mode: ammonia water, 70% of concentrated sugar and 5% of foam enemy;

and (3) fermentation period: about 18-20 h.

The result showed that the L-valine yield of Corynebacterium glutamicum CGMCC No.21260 was 84.1g/L, OD ₆₁₀ Is 98.2; the L-valine yield of YPV-097 is 85.7g/L, which is obviously higher than that of the strain CGMCC No.21260 (P)<0.05），OD ₆₁₀ Is 99.2; YPV-098 has L-valine yield of 87.6g/L, which is significantly higher than strain CGMCC No.21260 (P)<0.05），OD ₆₁₀ Was 100.3. Thus, it was shown that replacement of the ilvC original promoter with yfjB or EP6 promoter in the valine-producing bacterium CGMCC No.21260 increased the production of valine, and that the production of valine was higher as a mutant EP6 promoted the expression of ilvC.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is made possible within the scope of the claims attached below.

Sequence listing

<110> Beijing Zhongkou Yi Biotechnology Co., Ltd

<120> EP6 promoter, and related biological material and application thereof

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<213> Artificial sequence (Artificial sequence)

<400> 2

tctagagtcgacctgcaggcatgcaagcttggctgttttggcggatgagagaagattttc 60

agcctgatacagattaaatcagaacgcagaagcggtctgataaaacagaatttgcctggc 120

ggcagtagcgcggtggtcccacctgaccccatgccgaactcagaagtgaaacgccgtagc 180

gccgatggtagtgtggggtctccccatgcgagagtagggaactgccaggcatcaaataaa 240

acgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgc 300

tctcctgagtaggacaaatccgccgggagcggatttgaacgttgcgaagcaacggcccgg 360

agggtggcgggcaggacgcccgccataaactgccaggcatcaaattaagcagaaggccat 420

cctgacggatggcctttttgcgtttctacaaactctttttgtttatttttctaaatacat 480

tcaaatatgtatccgctcatgaattaattccgctagatgacgtgcggcttcgacctcctg 540

ggcgtggcgcttgttggcgcgctcgcggctggctgcggcacgacacgcgtctgagcagta 600

ttttgcgcgccgtcctcgtgggtcaggccggggtgggatcaggccaccgcagtaggcgca 660

gctgatgcgatcctccactactgcgcgtcctcctggcgctgccgagcacgcagctcgtcg 720

gccagctcttcaaggtcggccacaagcgtttctaggtcgctcgcggcacttgcccagtcg 780

cgtgatgctggcgcgtctgtcgtatcgagggcgcggaaaaatccgatcaccgtttttaaa 840

tcgacggcggcatcgagtgcgtcggactccagcgcgacatcggagagatccaccgctgat 900

gcttcaggccagttttggtacttcgtcgtgaaggtcatgacaccattataacgaacgttc 960

gttaaaaattctagccccaattctgataatttcttccggcactcctgcgaaaacctgcga 1020

gacttcttgcccagaaaaaacgccaagcgcagcggttaccgcactttttttccaggtgat 1080

ttcaccctgaccagcgaagcggcactttagtgcatgaggtgtgcccctggtttcccctct 1140

ttggagggttcaacccaaaaaagcacacaagcaaaaatgaaaatcatcatgagcaagttg 1200

gtgcgaagcagcaacgcgctagctccaaaaaggtctccaggatctcgaggagatttttga 1260

gggggagggagtcgaggaagagccagagcagaaggcggggaaccgttctctgccgacagc 1320

gtgagccccccttaaaaatcaggccggggaggaaccggggagggatcagagctaggagcg 1380

agacaccctaaagggggggaaccgttttctgctgacggtgtttcgtttattagttttcag 1440

cccgtggatagcggagggtgagggcaagtgagagccagagcaaggacgggacccctaaag 1500

gggggaaccgttttctgctgacggtgtttcgtttattagttttcagcccgtggacggccg 1560

cgtttagcttccattccaagtgcctttctgacttgttggatgcgcctttcactgacacct 1620

agttcgcctgcaagctcacgagtcgagggatcagcaaccgattgagaacgggcatccagg 1680

atcgcagttttgacgcgaagttcgagcaactcgcctgtcatttctcggcgtttgtttgct 1740

tccgctaatcgctgtcgcgtctcctgcgcatacttactttctgggtcagcccatctgcgt 1800

gcattcgatgtagctgcgccccgtcgccccatcgtcgctagagctttccgccctcggctg 1860

ctctgcgtttccacccgacgagcagggacgactggctggcctttagccacgtagccgcgc 1920

acacgacgcgccatcgtcaggcgatcacgcatggcgggaagatccggctcccggccgtct 1980

gcaccgaccgcctgggcaacgttgtacgccacttcatacgcgtcgatgatcttggcatct 2040

tttaggcgctcaccagcagctttgagctggtatcccacggtcaacgcgtggcgaaacgcg 2100

gtctcgtcgcgcgctcgctctggatttgtccagagcactcgcacgccgtcgatcaggtcg 2160

ccggacgcgtccagggcgctcggcaggctcgcgtccaaaatcgctagcgccttggcttct 2220

gcggtggcgcgttgtgccgcttcaatgcgggcgcgtccgctggaaaagtcctgctcaatg 2280

tactttttcggcttctgtgatccggtcatcgttcgagcaatctccattaggtcggccagc 2340

cgatccacacgatcatgctggcagtgccatttataggctgtcggatcgtctgagacgtgc 2400

agcggccaccggctcagcctatgcgaaaaagcctggtcagcgccgaaaacacgagtcatt 2460

tcttccgtcgttgcagccagcaggcgcatatttgggctggttttacctgctgcggcatac 2520

accgggtcaatgagccagatgagctggcatttcccgctcagcggattcacgccgatccaa 2580

gccggcgctttttctaggcgtgcccatttctctaaaatcgcgtagacctgcgggtttacg 2640

tgctcaatcttcccgccggcctggtggctgggcacatcgatgtcaagcacgatcaccgcg 2700

gcatgttgcgcgtgcgtcagcgcaacgtactggcaccgcgtcagcgcttttgagccagcc 2760

cggtagagctttggttgggtttcgccggtatccgggtttttaatccaggcgctcgcgaaa 2820

tctcttgtcttgctgccctggaagctttcgcgtcccaggtgagcgagcagttcgcggcga 2880

tcttctgccgtccagccgcgtgagccgcagcgcatagcttcggggtgggtgtcgaacaga 2940

tcggcggacaatttccacgcgctagctgtgactgtgtcctgcggatcggctagagtcatg 3000

tcttgagtgctttctcccagctgatgactgggggttagccgacgccctgtgagttcccgc 3060

tcacggggcgttcaactttttcaggtatttgtgcagcttatcgtgttttcttcgtaaatg 3120

aacgcttaactaccttgttaaacgtggcaaataggcaggattgatggggatctagcttca 3180

cgctgccgcaagcactcagggcgcaagggctgctaaaggaagcggaacacgtagaaagcc 3240

agtccgcagaaacggtgctgaccccggatgaatgtcagctactgggctatctggacaagg 3300

gaaaacgcaagcgcaaagagaaagcaggtagcttgcagtgggcttacatggcgatagcta 3360

gactgggcggttttatggacagcaagcgaaccggaattgccagctggggcgccctctggt 3420

aaggttgggaagccctgcaaagtaaactggatggctttcttgccgccaaggatctgatgg 3480

cgcaggggatcaagatctgatcaagagacaggatgaggatcgtttcgcatgattgaacaa 3540

gatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgg 3600

gcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgc 3660

ccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactccaagacgaggca 3720

gcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtc 3780

actgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtca 3840

tctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcat 3900

acgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagca 3960

cgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcagggg 4020

ctcgcgccagccgaactgttcgccaggctcaaggcgcggatgcccgacggcgaggatctc 4080

gtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttct 4140

ggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggct 4200

acccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttac 4260

ggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttc 4320

tgagcgggactctggggttcgcggaatcatgaccaaaatcccttaacgtgagttttcgtt 4380

ccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttct 4440

gcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgcc 4500

ggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagatacc 4560

aaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcacc 4620

gcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtc 4680

gtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctg 4740

aacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagata 4800

cctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggta 4860

tccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgc 4920

ctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtg 4980

atgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggtt 5040

cctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgt 5100

ggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccga 5160

gcgcagcgagtcagtgagcgaggaagcggaagagcgcctgatgcggtattttctccttac 5220

gcatctgtgcggtatttcacaccgcatatggtgcactctcagtacaatctgctctgatgc 5280

cgcatagttaagccagtatacactccgctatcgctacgtgactgggtcatggctgcgccc 5340

cgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgct 5400

tacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatca 5460

ccgaaacgcgcgaggcagcagatcaattcgcgcgcgaaggcgaagcggcatgcatttacg 5520

ttgacaccatcgaatggtgcaaaacctttcgcggtatggcatgatagcgcccggaagaga 5580

gtcaattcagggtggtgaatgtgaaaccagtaacgttatacgatgtcgcagagtatgccg 5640

gtgtctcttatcagaccgtttcccgcgtggtgaaccaggccagccacgtttctgcgaaaa 5700

cgcgggaaaaagtggaagcggcgatggcggagctgaattacattcccaaccgcgtggcac 5760

aacaactggcgggcaaacagtcgttgctgattggcgttgccacctccagtctggccctgc 5820

acgcgccgtcgcaaattgtcgcggcgattaaatctcgcgccgatcaactgggtgccagcg 5880

tggtggtgtcgatggtagaacgaagcggcgtcgaagcctgtaaagcggcggtgcacaatc 5940

ttctcgcgcaacgcgtcagtgggctgatcattaactatccgctggatgaccaggatgcca 6000

ttgctgtggaagctgcctgcactaatgttccggcgttatttcttgatgtctctgaccaga 6060

cacccatcaacagtattattttctcccatgaagacggtacgcgactgggcgtggagcatc 6120

tggtcgcattgggtcaccagcaaatcgcgctgttagcgggcccattaagttctgtctcgg 6180

cgcgtctgcgtctggctggctggcataaatatctcactcgcaatcaaattcagccgatag 6240

cggaacgggaaggcgactggagtgccatgtccggttttcaacaaaccatgcaaatgctga 6300

atgagggcatcgttcccactgcgatgctggttgccaacgatcagatggcgctgggcgcaa 6360

tgcgcgccattaccgagtccgggctgcgcgttggtgcggatatctcggtagtgggatacg 6420

acgataccgaagacagctcatgttatatcccgccgtcaaccaccatcaaacaggattttc 6480

gcctgctggggcaaaccagcgtggaccgcttgctgcaactctctcagggccaggcggtga 6540

agggcaatcagctgttgcccgtctcactggtgaaaagaaaaaccaccctggcgcccaata 6600

cgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggttt 6660

cccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagcgcgaattgatc 6720

tggtttgacagcttatcatcgactgcacggtgcaccaatgcttctggcgtcaggcagcca 6780

tcggaagctgtggtatggctgtgcaggtcgtaaatcactgcataattcgtgtcgctcaag 6840

gcgcactcccgttctggataatgttttttgcgccgacatcataacggttctggcaaatat 6900

tctgaaatgagctgttgacaattaatcatccggctcgtataatgtgtggaattgtgagcg 6960

gataacaatttcacacaggaaacagaccatggaattcgagctcggtacccggggatcctc 7020

taggaatttctccgcgtttttttcgcattcatctcgctaacttcgcttattatggggatc 7080

agtttcagggtttcaagggaagcactcacattgtcatcaatcttcgcaacaaggacctcg 7140

gaaaaatgcgtaaaggagaagaagataacatggctatcattaaagagttcatgcgcttca 7200

aagttcacatggagggttctgttaacggtcacgagttcgagatcgaaggcgaaggcgagg 7260

gccgtccgtatgaaggcacccagaccgccaaactgaaagtgactaaaggcggcccgctgc 7320

cttttgcgtgggacatcctgagcccgcaatttatgtacggttctaaagcgtatgttaaac 7380

acccagcggatatcccggactatctgaagctgtcttttccggaaggtttcaagtgggaac 7440

gcgtaatgaattttgaagatggtggtgtcgtgaccgtcactcaggactcctccctgcagg 7500

atggcgagttcatctataaagttaaactgcgtggtactaattttccatctgatggcccgg 7560

tgatgcagaaaaagacgatgggttgggaggcgtctagcgaacgcatgtatccggaagatg 7620

gtgcgctgaaaggcgaaattaaacagcgcctgaaactgaaagatggcggccattatgacg 7680

ctgaagtgaaaaccacgtacaaagccaagaaacctgtgcagctgcctggcgcgtacaatg 7740

tgaatattaaactggacatcacctctcataatgaagattatacgatcgtagagcaatatg 7800

agcgcgcggagggtcgtcattctaccggtggcatggatgaactatacaaactagagtcga 7860

cctgcaggcatgcaagcttggctgttttggcgga 7894

<210> 3

<211> 124

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 3

gaatttcttcgcgttttttttgcatccatcttgttaacttcgcttattatggggatcagt 60

ttccgggtttcaagggaagcggacacattgtcactatttcatttaacgcagggacctcca 120

aatc 124

<210> 4

<211> 1486

<212> DNA

<213> Corynebacterium glutamicum (Corynebacterium glutamicum)

<400> 4

tacggctccaaggacgtttgttttctgggtcagttaccccaaaaagcatatacagagacc 60

aatgatttttcattaaaaaggcagggatttgttataagtatgggtcgtattctgtgcgac 120

gggtgtacctcggctagaatttctccccatggctacagttgaaaatttcaatgaacttcc 180

cgcacacgtatggccacgcaatgccgtgcgccaagaagacggcgttgtcaccgtcgctgg 240

tgtgcctctgcctgacctcgctgaagaatacggaaccccactgttcgtagtcgacgagga 300

cgatttccgttcccgctgtcgcgacatggctaccgcattcggtggaccaggcaatgtgca 360

ctacgcatctaaagcgttcctgaccaagaccattgcacgttgggttgatgaagaggggct 420

ggcactggacattgcatccatcaacgaactgggcattgccctggccgctggtttccccgc 480

cagccgtatcaccgcgcacggcaacaacaaaggcgtagagttcctgcgcgcgttggttca 540

aaacggtgtgggacacgtggtgctggactccgcacaggaactagaactgttggattacgt 600

tgccgctggtgaaggcaagattcaggacgtgttgatccgcgtaaagccaggcatcgaagc 660

acacacccacgagttcatcgccactagccacgaagaccagaagttcggattctccctggc 720

atccggttccgcattcgaagcagcaaaagccgccaacaacgcagaaaacctgaacctggt 780

tggcctgcactgccacgttggttcccaggtgttcgacgccgaaggcttcaagctggcagc 840

agaacgcgtgttgggcctgtactcacagatccacagcgaactgggcgttgcccttcctga 900

actggatctcggtggcggatacggcattgcctataccgcagctgaagaaccactcaacgt 960

cgcagaagttgcctccgacctgctcaccgcagtcggaaaaatggcagcggaactaggcat 1020

cgacgcaccaaccgtgcttgttgagcccggccgcgctatcgcaggcccctccaccgtgac 1080

catctacgaagtcggcaccaccaaagacgtccacgtagacgacgacaaaacccgccgtta 1140

catcgccgtggacggaggcatgtccgacaacatccgcccagcactctacggctccgaata 1200

cgacgcccgcgtagtatcccgcttcgccgaaggagacccagtaagcacccgcatcgtggg 1260

ctcccactgcgaatccggcgatatcctgatcaacgatgaaatctacccatctgacatcac 1320

cagcggcgacttccttgcactcgcagccaccggcgcatactgctacgccatgagctcccg 1380

ctacaacgccttcacacggcccgccgtcgtgtccgtccgcgctggcagctcccgcctcat 1440

gctgcgccgcgaaacgctcgacgacatcctctcactagaggcataa 1486

<210> 5

<211> 1648

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 5

cagtgccaagcttgcatgcctgcaggtcgactctagcccaggtgaagaagtcgttgaggg 60

agtttttgtagccctgttcttgggaggccatgatgccgttccatgccaggctgggcgcca 120

ttgcggaatcgaggacaaccttgtcggtgtgctgtgggtagcgggtggcgtagaccgatc 180

cgaggtaggttccgtaggacagtccgaagatggagatcttgtcatcgccaagtgcttggc 240

ggacgcgctcccagtcgttggcggtgttgtcggtggtcaggctggaggtgtagccggggg 300

tgccgatctcgcaggattctttaacgaaagcgccttcgcgggtgagcagcgagaggaaat 360

cgtatcctggtgcgatgttgtcgcagttaaccggtgtggagccgaccattccgcgaggct 420

gcactgcaacgaggtcgtagttttggtacatggcttctggccagttcatggattggctgc 480

cgaagaagctataggcatcgccaccagggccaccggagttaccgaagatggtgccgtgct 540

tttcgccttgggcagggaccttgacaaagcccacgctgatatcgccaagtgagggatcag 600

aatagtgcatgggcacgtcgatgctgccacattgagcggaggcaatatctacctgaggtg 660

ggcattcttcccagcggatgttttcttgcgctgctgcagtgggcattgataccaaaaagg 720

ggctaagcgcagtcgaggcggcaagaactgctactaccctttttattgtcgaacggggca 780

tgaatttctccgcgtttttttcgcattcatctcgctaacttcgcttattatggggatcag 840

tttcagggtttcaagggaagcactcacattgtcatcaatcttcgcaacaaggacctcgga 900

aaaatgacaccagctgatctcgcaacattgattaaagagaccgcggtagaggttttgacc 960

tcccgcgagctcgatacttctgttcttccggagcaggtagttgtggagcgtccgcgtaac 1020

ccagagcacggcgattacgccaccaacattgcattgcaggtggctaaaaaggtcggtcag 1080

aaccctcgggatttggctacctggctggcagaggcattggctgcagatgacgccattgat 1140

tctgctgaaattgctggcccaggctttttgaacattcgccttgctgcagcagcacagggt 1200

gaaattgtggccaagattctggcacagggcgagactttcggaaactccgatcacctttcc 1260

cacttggacgtgaacctcgagttcgtttctgcaaacccaaccggacctattcaccttggc 1320

ggaacccgctgggctgccgtgggtgactctttgggtcgtgtgctggaggcttccggcgcg 1380

aaagtgacccgcgaatactacttcaacgatcacggtcgccagatcgatcgtttcgctttg 1440

tcccttcttgcagcggcgaagggcgagccaacgccagaagacggttatggcggcgaatac 1500

attaaggaaattgcggaggcaatcgtcgaaaagcatcctgaagcgttggctttggagcct 1560

gccgcaacccaggagcttttccgcgctgaaggcgtggagatgatgttcgagggtaccgag 1620

ctcgaattcgtaatcatggtcatagctg 1648

<210> 6

<211> 1650

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 6

cagtgccaagcttgcatgcctgcaggtcgactctagcccaggtgaagaagtcgttgaggg 60

agtttttgtagccctgttcttgggaggccatgatgccgttccatgccaggctgggcgcca 120

ttgcggaatcgaggacaaccttgtcggtgtgctgtgggtagcgggtggcgtagaccgatc 180

cgaggtaggttccgtaggacagtccgaagatggagatcttgtcatcgccaagtgcttggc 240

ggacgcgctcccagtcgttggcggtgttgtcggtggtcaggctggaggtgtagccggggg 300

tgccgatctcgcaggattctttaacgaaagcgccttcgcgggtgagcagcgagaggaaat 360

cgtatcctggtgcgatgttgtcgcagttaaccggtgtggagccgaccattccgcgaggct 420

gcactgcaacgaggtcgtagttttggtacatggcttctggccagttcatggattggctgc 480

cgaagaagctataggcatcgccaccagggccaccggagttaccgaagatggtgccgtgct 540

tttcgccttgggcagggaccttgacaaagcccacgctgatatcgccaagtgagggatcag 600

aatagtgcatgggcacgtcgatgctgccacattgagcggaggcaatatctacctgaggtg 660

ggcattcttcccagcggatgttttcttgcgctgctgcagtgggcattgataccaaaaagg 720

ggctaagcgcagtcgaggcggcaagaactgctactaccctttttattgtcgaacggggca 780

tgaatttcttcgcgttttttttgcatccatcttgttaacttcgcttattatggggatcag 840

tttccgggtttcaagggaagcggacacattgtcactatttcatttaacgcagggacctcc 900

aaatcatgacaccagctgatctcgcaacattgattaaagagaccgcggtagaggttttga 960

cctcccgcgagctcgatacttctgttcttccggagcaggtagttgtggagcgtccgcgta 1020

acccagagcacggcgattacgccaccaacattgcattgcaggtggctaaaaaggtcggtc 1080

agaaccctcgggatttggctacctggctggcagaggcattggctgcagatgacgccattg 1140

attctgctgaaattgctggcccaggctttttgaacattcgccttgctgcagcagcacagg 1200

gtgaaattgtggccaagattctggcacagggcgagactttcggaaactccgatcaccttt 1260

cccacttggacgtgaacctcgagttcgtttctgcaaacccaaccggacctattcaccttg 1320

gcggaacccgctgggctgccgtgggtgactctttgggtcgtgtgctggaggcttccggcg 1380

cgaaagtgacccgcgaatactacttcaacgatcacggtcgccagatcgatcgtttcgctt 1440

tgtcccttcttgcagcggcgaagggcgagccaacgccagaagacggttatggcggcgaat 1500

acattaaggaaattgcggaggcaatcgtcgaaaagcatcctgaagcgttggctttggagc 1560

ctgccgcaacccaggagcttttccgcgctgaaggcgtggagatgatgttcgagggtaccg 1620

agctcgaattcgtaatcatggtcatagctg 1650

<210> 7

<211> 885

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 7

ggcgacgatcttgtttgaccagttttggtacacggctagtggggtagtgccgaggccata 60

cgtgtcgttgttttctgcaacccaggtgaagaagtcgttgagggagtttttgtagccctg 120

ttcttgggaggccatgatgccgttccatgccaggctgggcgccattgcggaatcgaggac 180

aaccttgtcggtgtgctgtgggtagcgggtggcgtagaccgatccgaggtaggttccgta 240

ggacagtccgaagatggagatcttgtcatcgccaagtgcttggcggacgcgctcccagtc 300

gttggcggtgttgtcggtggtcaggctggaggtgtagccgggggtgccgatctcgcagga 360

ttctttaacgaaagcgccttcgcgggtgagcagcgagaggaaatcgtatcctggtgcgat 420

gttgtcgcagttaaccggtgtggagccgaccattccgcgaggctgcactgcaacgaggtc 480

gtagttttggtacatggcttctggccagttcatggattggctgccgaagaagctataggc 540

atcgccaccagggccaccggagttaccgaagatggtgccgtgcttttcgccttgggcagg 600

gaccttgacaaagcccacgctgatatcgccaagtgagggatcagaatagtgcatgggcac 660

gtcgatgctgccacattgagcggaggcaatatctacctgaggtgggcattcttcccagcg 720

gatgttttcttgcgctgctgcagtgggcattgataccaaaaaggggctaagcgcagtcga 780

ggcggcaagaactgctactaccctttttattgtcgaacggggcatgaatttctccgcgtt 840

tttttcgcattcatctcgctaacttcgcttattatggggatcagt 885

<210> 8

<211> 885

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 8

ggcgacgatcttgtttgaccagttttggtacacggctagtggggtagtgccgaggccata 60

cgtgtcgttgttttctgcaacccaggtgaagaagtcgttgagggagtttttgtagccctg 120

ttcttgggaggccatgatgccgttccatgccaggctgggcgccattgcggaatcgaggac 180

aaccttgtcggtgtgctgtgggtagcgggtggcgtagaccgatccgaggtaggttccgta 240

ggacagtccgaagatggagatcttgtcatcgccaagtgcttggcggacgcgctcccagtc 300

gttggcggtgttgtcggtggtcaggctggaggtgtagccgggggtgccgatctcgcagga 360

ttctttaacgaaagcgccttcgcgggtgagcagcgagaggaaatcgtatcctggtgcgat 420

gttgtcgcagttaaccggtgtggagccgaccattccgcgaggctgcactgcaacgaggtc 480

gtagttttggtacatggcttctggccagttcatggattggctgccgaagaagctataggc 540

atcgccaccagggccaccggagttaccgaagatggtgccgtgcttttcgccttgggcagg 600

gaccttgacaaagcccacgctgatatcgccaagtgagggatcagaatagtgcatgggcac 660

gtcgatgctgccacattgagcggaggcaatatctacctgaggtgggcattcttcccagcg 720

gatgttttcttgcgctgctgcagtgggcattgataccaaaaaggggctaagcgcagtcga 780

ggcggcaagaactgctactaccctttttattgtcgaacggggcatgaatttcttcgcgtt 840

ttttttgcatccatcttgttaacttcgcttattatggggatcagt 885

<210> 9

<211> 841

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 9

cgcttattatggggatcagtttcagggtttcaagggaagcactcacattgtcatcaatct 60

tcgcaacaaggacctcggaaaaatgacaccagctgatctcgcaacattgattaaagagac 120

cgcggtagaggttttgacctcccgcgagctcgatacttctgttcttccggagcaggtagt 180

tgtggagcgtccgcgtaacccagagcacggcgattacgccaccaacattgcattgcaggt 240

ggctaaaaaggtcggtcagaaccctcgggatttggctacctggctggcagaggcattggc 300

tgcagatgacgccattgattctgctgaaattgctggcccaggctttttgaacattcgcct 360

tgctgcagcagcacagggtgaaattgtggccaagattctggcacagggcgagactttcgg 420

aaactccgatcacctttcccacttggacgtgaacctcgagttcgtttctgcaaacccaac 480

cggacctattcaccttggcggaacccgctgggctgccgtgggtgactctttgggtcgtgt 540

gctggaggcttccggcgcgaaagtgacccgcgaatactacttcaacgatcacggtcgcca 600

gatcgatcgtttcgctttgtcccttcttgcagcggcgaagggcgagccaacgccagaaga 660

cggttatggcggcgaatacattaaggaaattgcggaggcaatcgtcgaaaagcatcctga 720

agcgttggctttggagcctgccgcaacccaggagcttttccgcgctgaaggcgtggagat 780

gatgttcgagcacatcaaatcttccctgcatgagttcggcaccgatttcgatgtctacta 840

c 841

<210> 10

<211> 843

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 10

cgcttattatggggatcagtttccgggtttcaagggaagcggacacattgtcactatttc 60

atttaacgcagggacctccaaatcatgacaccagctgatctcgcaacattgattaaagag 120

accgcggtagaggttttgacctcccgcgagctcgatacttctgttcttccggagcaggta 180

gttgtggagcgtccgcgtaacccagagcacggcgattacgccaccaacattgcattgcag 240

gtggctaaaaaggtcggtcagaaccctcgggatttggctacctggctggcagaggcattg 300

gctgcagatgacgccattgattctgctgaaattgctggcccaggctttttgaacattcgc 360

cttgctgcagcagcacagggtgaaattgtggccaagattctggcacagggcgagactttc 420

ggaaactccgatcacctttcccacttggacgtgaacctcgagttcgtttctgcaaaccca 480

accggacctattcaccttggcggaacccgctgggctgccgtgggtgactctttgggtcgt 540

gtgctggaggcttccggcgcgaaagtgacccgcgaatactacttcaacgatcacggtcgc 600

cagatcgatcgtttcgctttgtcccttcttgcagcggcgaagggcgagccaacgccagaa 660

gacggttatggcggcgaatacattaaggaaattgcggaggcaatcgtcgaaaagcatcct 720

gaagcgttggctttggagcctgccgcaacccaggagcttttccgcgctgaaggcgtggag 780

atgatgttcgagcacatcaaatcttccctgcatgagttcggcaccgatttcgatgtctac 840

tac 843

<210> 11

<211> 1161

<212> DNA

<213> Corynebacterium glutamicum (Corynebacterium glutamicum)

<400> 11

ccccctctttaaggacagaaaggtttcaaccaagacatgttcgagctaatcgacgactgg 60

ggtcccgaaaagatcgtcatcgtcagcgatcaaaaaaccgggatgcgtggcgtacttgtc 120

atcgacaacaccgcccgcggcatgggcaagggcggcacgcgcatgcagcccaccgtttca 180

gtcgcagaaatagccaggttggctcgcgtcatgacctggaaatgggctggtgtagacctc 240

ttttatggtggtgcaaaagccggaatccaagcagaccccacctccccagataaagaagca 300

atccttcggtcattcgtcagaaaactctccaacgaagtacctaaagaatatgtcttcggc 360

ctggacatggggctgactgaaaatgacgccgccatcatcgtcgacgagctcggctggggc 420

accagtatgggaacaccctacgagctcggtggagtgccctacgacaagcttggtatcacc 480

ggctttggtgttgcagaagtggtggatcaagtagcacaaatgcaaaaactcaaaggtgca 540

tcggtagcagtccaaggcttcggtgccgttggacatgccacagcttcccgcctggcagaa 600

cttggctatcctgttgtggctatctccacagcaaagggagcaatcgcagaccccaacggg 660

ctcaacatccccgagctcatggaactacgcgatcaggtgggtgactcacttgtggaccac 720

tacccagcacttcgcatcaacccaggtgacgaacttttcaccgaagccgaaatcctcgta 780

ccggcagcgctccaggacgtcatcgatgaagacgcagccaatcgactacaagcacaactc 840

gtcgtcgaaggagcaaaccttcccactaatgaagccgcacaaaaagtcctaagtaaccgt 900

ggcatcactgtggttccagactttgtcgccaacgccggaggagtagtcgccgcagcattc 960

gccatggataaccgcatgtctgcattccgtgcagagactgccaacatcttcacgagcgtt 1020

tccgacaaactccgatccaatgccgaaactgtcctgaatttcacctctgaatctgacctg 1080

accagccacgaagcggcaaggcaactttcacaggaacgagttcttgcagctatgcgcgcc 1140

agaggtatgg ttcgccgata a 1161

<210> 12

<211> 1699

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 12

cagtgccaagcttgcatgcctgcaggtcgactctagcggaaacaacgcagccatagtcac 60

cccagatgcagatcttgacctggcacttcgcggaattgtcttcgcggctgccggaaccgc 120

tggtcagcgctgcaccaccatgcgccgcgttatcgtccacgagtccattgcagaagagct 180

caccgaaaaactcgtttccgcataccaaaccctgaccatcggcgatcctcgcgacgagca 240

gatcctcgttggaccactgatcaatgaatccggattccagggtatgcaggatgcactcaa 300

ggctgcaaccgaacagggcggaacggttatcaccggtggcaaccgagtcctcgaagatga 360

attcccagaggcctactacgttgagcctgcaatcgtaaccatgccagcacaaacagacat 420

tgtgcgcgatgaaaccttcgcaccaattctctacgtgctgacctactccactttggaaga 480

agcaatcgcactccaaaacgatgttccacagggcctttcttctgcaatcttcaccgaaaa 540

ccagcgcgaagcagagctcttcgtctccgcttccggctccgactgtggcattgccaacgt 600

caacatcggtacctccggcgcagaaatcggcggcgcattcggtggcgaaaaggaaaccgg 660

tggcggacgcgaatccggctccgactcctggaagtcctacatgcgacgcgccaccaacac 720

cgtcaactactccggcgaactgccactcgcccaaggcgttaaattcctctaagaatttct 780

ccgcgtttttttcgcattcatctcgctaacttcgcttattatggggatcagtttcagggt 840

ttcaagggaagcactcacattgtcatcaatcttcgcaacaaggacctcggaaaaatgttc 900

gagctaatcgacgactggggtcccgaaaagatcgtcatcgtcagcgatcaaaaaaccggg 960

atgcgtggcgtacttgtcatcgacaacaccgcccgcggcatgggcaagggcggcacgcgc 1020

atgcagcccaccgtttcagtcgcagaaatagccaggttggctcgcgtcatgacctggaaa 1080

tgggctggtgtagacctcttttatggtggtgcaaaagccggaatccaagcagaccccacc 1140

tccccagataaagaagcaatccttcggtcattcgtcagaaaactctccaacgaagtacct 1200

aaagaatatgtcttcggcctggacatggggctgactgaaaatgacgccgccatcatcgtc 1260

gacgagctcggctggggcaccagtatgggaacaccctacgagctcggtggagtgccctac 1320

gacaagcttggtatcaccggctttggtgttgcagaagtggtggatcaagtagcacaaatg 1380

caaaaactcaaaggtgcatcggtagcagtccaaggcttcggtgccgttggacatgccaca 1440

gcttcccgcctggcagaacttggctatcctgttgtggctatctccacagcaaagggagca 1500

atcgcagaccccaacgggctcaacatccccgagctcatggaactacgcgatcaggtgggt 1560

gactcacttgtggaccactacccagcacttcgcatcaacccaggtgacgaacttttcacc 1620

gaagccgaaatcctcgtaccggcagcgctccaggacgtcatgggtaccgagctcgaattc 1680

gtaatcatgg tcatagctg 1699

<210> 13

<211> 1701

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 13

cagtgccaagcttgcatgcctgcaggtcgactctagcggaaacaacgcagccatagtcac 60

cccagatgcagatcttgacctggcacttcgcggaattgtcttcgcggctgccggaaccgc 120

tggtcagcgctgcaccaccatgcgccgcgttatcgtccacgagtccattgcagaagagct 180

caccgaaaaactcgtttccgcataccaaaccctgaccatcggcgatcctcgcgacgagca 240

gatcctcgttggaccactgatcaatgaatccggattccagggtatgcaggatgcactcaa 300

ggctgcaaccgaacagggcggaacggttatcaccggtggcaaccgagtcctcgaagatga 360

attcccagaggcctactacgttgagcctgcaatcgtaaccatgccagcacaaacagacat 420

tgtgcgcgatgaaaccttcgcaccaattctctacgtgctgacctactccactttggaaga 480

agcaatcgcactccaaaacgatgttccacagggcctttcttctgcaatcttcaccgaaaa 540

ccagcgcgaagcagagctcttcgtctccgcttccggctccgactgtggcattgccaacgt 600

caacatcggtacctccggcgcagaaatcggcggcgcattcggtggcgaaaaggaaaccgg 660

tggcggacgcgaatccggctccgactcctggaagtcctacatgcgacgcgccaccaacac 720

cgtcaactactccggcgaactgccactcgcccaaggcgttaaattcctctaagaatttct 780

tcgcgttttttttgcatccatcttgttaacttcgcttattatggggatcagtttccgggt 840

ttcaagggaagcggacacattgtcactatttcatttaacgcagggacctccaaatcatgt 900

tcgagctaatcgacgactggggtcccgaaaagatcgtcatcgtcagcgatcaaaaaaccg 960

ggatgcgtggcgtacttgtcatcgacaacaccgcccgcggcatgggcaagggcggcacgc 1020

gcatgcagcccaccgtttcagtcgcagaaatagccaggttggctcgcgtcatgacctgga 1080

aatgggctggtgtagacctcttttatggtggtgcaaaagccggaatccaagcagacccca 1140

cctccccagataaagaagcaatccttcggtcattcgtcagaaaactctccaacgaagtac 1200

ctaaagaatatgtcttcggcctggacatggggctgactgaaaatgacgccgccatcatcg 1260

tcgacgagctcggctggggcaccagtatgggaacaccctacgagctcggtggagtgccct 1320

acgacaagcttggtatcaccggctttggtgttgcagaagtggtggatcaagtagcacaaa 1380

tgcaaaaactcaaaggtgcatcggtagcagtccaaggcttcggtgccgttggacatgcca 1440

cagcttcccgcctggcagaacttggctatcctgttgtggctatctccacagcaaagggag 1500

caatcgcagaccccaacgggctcaacatccccgagctcatggaactacgcgatcaggtgg 1560

gtgactcacttgtggaccactacccagcacttcgcatcaacccaggtgacgaacttttca 1620

ccgaagccgaaatcctcgtaccggcagcgctccaggacgtcatgggtaccgagctcgaat 1680

tcgtaatcatggtcatagct g 1701

<210> 14

<211> 849

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 14

gaagtaggtcctcgtgttgctgcccgattcggcaaaaccatcctggagctgggcggaaac 60

aacgcagccatagtcaccccagatgcagatcttgacctggcacttcgcggaattgtcttc 120

gcggctgccggaaccgctggtcagcgctgcaccaccatgcgccgcgttatcgtccacgag 180

tccattgcagaagagctcaccgaaaaactcgtttccgcataccaaaccctgaccatcggc 240

gatcctcgcgacgagcagatcctcgttggaccactgatcaatgaatccggattccagggt 300

atgcaggatgcactcaaggctgcaaccgaacagggcggaacggttatcaccggtggcaac 360

cgagtcctcgaagatgaattcccagaggcctactacgttgagcctgcaatcgtaaccatg 420

ccagcacaaacagacattgtgcgcgatgaaaccttcgcaccaattctctacgtgctgacc 480

tactccactttggaagaagcaatcgcactccaaaacgatgttccacagggcctttcttct 540

gcaatcttcaccgaaaaccagcgcgaagcagagctcttcgtctccgcttccggctccgac 600

tgtggcattgccaacgtcaacatcggtacctccggcgcagaaatcggcggcgcattcggt 660

ggcgaaaaggaaaccggtggcggacgcgaatccggctccgactcctggaagtcctacatg 720

cgacgcgccaccaacaccgtcaactactccggcgaactgccactcgcccaaggcgttaaa 780

ttcctctaagaatttctccgcgtttttttcgcattcatctcgctaacttcgcttattatg 840

gggatcagt 849

<210> 15

<211> 849

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 15

gaagtaggtcctcgtgttgctgcccgattcggcaaaaccatcctggagctgggcggaaac 60

aacgcagccatagtcaccccagatgcagatcttgacctggcacttcgcggaattgtcttc 120

gcggctgccggaaccgctggtcagcgctgcaccaccatgcgccgcgttatcgtccacgag 180

tccattgcagaagagctcaccgaaaaactcgtttccgcataccaaaccctgaccatcggc 240

gatcctcgcgacgagcagatcctcgttggaccactgatcaatgaatccggattccagggt 300

atgcaggatgcactcaaggctgcaaccgaacagggcggaacggttatcaccggtggcaac 360

cgagtcctcgaagatgaattcccagaggcctactacgttgagcctgcaatcgtaaccatg 420

ccagcacaaacagacattgtgcgcgatgaaaccttcgcaccaattctctacgtgctgacc 480

tactccactttggaagaagcaatcgcactccaaaacgatgttccacagggcctttcttct 540

gcaatcttcaccgaaaaccagcgcgaagcagagctcttcgtctccgcttccggctccgac 600

tgtggcattgccaacgtcaacatcggtacctccggcgcagaaatcggcggcgcattcggt 660

ggcgaaaaggaaaccggtggcggacgcgaatccggctccgactcctggaagtcctacatg 720

cgacgcgccaccaacaccgtcaactactccggcgaactgccactcgcccaaggcgttaaa 780

ttcctctaagaatttcttcgcgttttttttgcatccatcttgttaacttcgcttattatg 840

gggatcagt 849

<210> 16

<211> 889

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 16

cgcttattatggggatcagtttcagggtttcaagggaagcactcacattgtcatcaatct 60

tcgcaacaaggacctcggaaaaatgttcgagctaatcgacgactggggtcccgaaaagat 120

cgtcatcgtcagcgatcaaaaaaccgggatgcgtggcgtacttgtcatcgacaacaccgc 180

ccgcggcatgggcaagggcggcacgcgcatgcagcccaccgtttcagtcgcagaaatagc 240

caggttggctcgcgtcatgacctggaaatgggctggtgtagacctcttttatggtggtgc 300

aaaagccggaatccaagcagaccccacctccccagataaagaagcaatccttcggtcatt 360

cgtcagaaaactctccaacgaagtacctaaagaatatgtcttcggcctggacatggggct 420

gactgaaaatgacgccgccatcatcgtcgacgagctcggctggggcaccagtatgggaac 480

accctacgagctcggtggagtgccctacgacaagcttggtatcaccggctttggtgttgc 540

agaagtggtggatcaagtagcacaaatgcaaaaactcaaaggtgcatcggtagcagtcca 600

aggcttcggtgccgttggacatgccacagcttcccgcctggcagaacttggctatcctgt 660

tgtggctatctccacagcaaagggagcaatcgcagaccccaacgggctcaacatccccga 720

gctcatggaactacgcgatcaggtgggtgactcacttgtggaccactacccagcacttcg 780

catcaacccaggtgacgaacttttcaccgaagccgaaatcctcgtaccggcagcgctcca 840

ggacgtcatcgatgaagacgcagccaatcgactacaagcacaactcgtc 889

<210> 17

<211> 891

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 17

cgcttattatggggatcagtttccgggtttcaagggaagcggacacattgtcactatttc 60

atttaacgcagggacctccaaatcatgttcgagctaatcgacgactggggtcccgaaaag 120

atcgtcatcgtcagcgatcaaaaaaccgggatgcgtggcgtacttgtcatcgacaacacc 180

gcccgcggcatgggcaagggcggcacgcgcatgcagcccaccgtttcagtcgcagaaata 240

gccaggttggctcgcgtcatgacctggaaatgggctggtgtagacctcttttatggtggt 300

gcaaaagccggaatccaagcagaccccacctccccagataaagaagcaatccttcggtca 360

ttcgtcagaaaactctccaacgaagtacctaaagaatatgtcttcggcctggacatgggg 420

ctgactgaaaatgacgccgccatcatcgtcgacgagctcggctggggcaccagtatggga 480

acaccctacgagctcggtggagtgccctacgacaagcttggtatcaccggctttggtgtt 540

gcagaagtggtggatcaagtagcacaaatgcaaaaactcaaaggtgcatcggtagcagtc 600

caaggcttcggtgccgttggacatgccacagcttcccgcctggcagaacttggctatcct 660

gttgtggctatctccacagcaaagggagcaatcgcagaccccaacgggctcaacatcccc 720

gagctcatggaactacgcgatcaggtgggtgactcacttgtggaccactacccagcactt 780

cgcatcaacccaggtgacgaacttttcaccgaagccgaaatcctcgtaccggcagcgctc 840

caggacgtcatcgatgaagacgcagccaatcgactacaagcacaactcgt c 891

<210> 18

<211> 1196

<212> DNA

<213> Corynebacterium glutamicum (Corynebacterium glutamicum)

<400> 18

acagcaattaatctgattgcacctgctgcataaatgtgactagtcaaacaccgtctaatt 60

gcatgtgtgtggtagaacaataatgtagttgtctgcccaagcgagtttaactcccacgat 120

ttacagtgggggcagacatcttttcaccaaaatttttacgaaaggcgagattttctccca 180

tggctattgaactgctttatgatgctgacgctgacctctccttgatccagggccgcaagg 240

ttgccatcgttggctacggctcccagggccacgcacactcccagaacctccgcgattctg 300

gcgttgaggttgtcattggtctgcgcgagggctccaagtccgcagagaaggcaaaggaag 360

caggcttcgaggtcaagaccaccgctgaggctgcagcttgggctgacgtcatcatgctcc 420

tggctccagacacctcccaggcagaaatcttcaccaacgacatcgagccaaacctgaacg 480

caggcgacgcactgctgttcggccacggcctgaacattcacttcgacctgatcaagccag 540

ctgacgacatcatcgttggcatggttgcgccaaagggcccaggccacttggttcgccgtc 600

agttcgttgatggcaagggtgttccttgcctcatcgcagtcgaccaggacccaaccggaa 660

ccgcacaggctctgaccctgtcctacgcagcagcaatcggtggcgcacgcgcaggcgtta 720

tcccaaccaccttcgaagctgagaccgtcaccgacctcttcggcgagcaggctgttctct 780

gcggtggcaccgaggaactggtcaaggttggcttcgaggttctcaccgaagctggctacg 840

agccagagatggcatacttcgaggttcttcacgagctcaagctcatcgttgacctcatgt 900

tcgaaggtggcatcagcaacatgaactactctgtttctgacaccgctgagttcggtggct 960

acctctccggcccacgcgtcatcgatgcagacaccaagtcccgcatgaaggacatcctga 1020

ccgatatccaggacggcaccttcaccaagcgcctcatcgcaaacgttgagaacggcaaca 1080

ccgaacttgagggccttcgtgcttcctacaacaaccacccaatcgaggagaccggcgcta 1140

agctccgcgacctcatgagctgggtcaaggttgacgctcgcgcagaaaccgcttaa 1196

<210> 19

<211> 1822

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 19

cagtgccaagcttgcatgcctgcaggtcgactctagagagatcaacgaccgcccagtagt 60

catcgacttcatcgtcggtgaagacgcacaggtatggccaatggtgtctgctggatcatc 120

caactccgatatccagtacgcactcggattgcgcccattctttgatggtgatgaatctgc 180

agcagaagatcctgccgacattcacgaagccgtcagcgacattgatgccgccgttgaatc 240

gaccgaggcataaggagagacccaagatggctaattctgacgtcacccgccacatcctgt 300

ccgtactcgttcaggacgtagacggaatcatttcccgcgtatcaggtatgttcacccgac 360

gcgcattcaacctcgtgtccctcgtgtctgcaaagaccgaaacactcggcatcaaccgca 420

tcacggttgttgtcgacgccgacgagctcaacattgagcagatcaccaagcagctcaaca 480

agctgatccccgtgctcaaagtcgtgcgacttgatgaagagaccaccatcgcccgcgcaa 540

tcatgctggttaaggtctctgcggatagcaccaaccgtccgcagatcgtcgacgccgcga 600

acatcttccgcgcccgagtcgtcgacgtggctccagactctgtggttattgaatccacag 660

gcaccccaggcaagctccgcgcactgcttgatgtgatggaaccattcggaatccgcgaac 720

tgatccaatccggacagattgcactcaaccgcggtccgaagaccatggctccggccaaga 780

tctaagaatttctccgcgtttttttcgcattcatctcgctaacttcgcttattatgggga 840

tcagtttcagggtttcaagggaagcactcacattgtcatcaatcttcgcaacaaggacct 900

cggaaaaatggctattgaactgctttatgatgctgacgctgacctctccttgatccaggg 960

ccgcaaggttgccatcgttggctacggctcccagggccacgcacactcccagaacctccg 1020

cgattctggcgttgaggttgtcattggtctgcgcgagggctccaagtccgcagagaaggc 1080

aaaggaagcaggcttcgaggtcaagaccaccgctgaggctgcagcttgggctgacgtcat 1140

catgctcctggctccagacacctcccaggcagaaatcttcaccaacgacatcgagccaaa 1200

cctgaacgcaggcgacgcactgctgttcggccacggcctgaacattcacttcgacctgat 1260

caagccagctgacgacatcatcgttggcatggttgcgccaaagggcccaggccacttggt 1320

tcgccgtcagttcgttgatggcaagggtgttccttgcctcatcgcagtcgaccaggaccc 1380

aaccggaaccgcacaggctctgaccctgtcctacgcagcagcaatcggtggcgcacgcgc 1440

aggcgttatcccaaccaccttcgaagctgagaccgtcaccgacctcttcggcgagcaggc 1500

tgttctctgcggtggcaccgaggaactggtcaaggttggcttcgaggttctcaccgaagc 1560

tggctacgagccagagatggcatacttcgaggttcttcacgagctcaagctcatcgttga 1620

cctcatgttcgaaggtggcatcagcaacatgaactactctgtttctgacaccgctgagtt 1680

cggtggctacctctccggcccacgcgtcatcgatgcagacaccaagtcccgcatgaagga 1740

catcctgaccgatatccaggacggcaccttcaccaagcgcctcagggtaccgagctcgaa 1800

ttcgtaatcatggtcatagctg 1822

<210> 20

<211> 1824

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 20

cagtgccaagcttgcatgcctgcaggtcgactctagagagatcaacgaccgcccagtagt 60

catcgacttcatcgtcggtgaagacgcacaggtatggccaatggtgtctgctggatcatc 120

caactccgatatccagtacgcactcggattgcgcccattctttgatggtgatgaatctgc 180

agcagaagatcctgccgacattcacgaagccgtcagcgacattgatgccgccgttgaatc 240

gaccgaggcataaggagagacccaagatggctaattctgacgtcacccgccacatcctgt 300

ccgtactcgttcaggacgtagacggaatcatttcccgcgtatcaggtatgttcacccgac 360

gcgcattcaacctcgtgtccctcgtgtctgcaaagaccgaaacactcggcatcaaccgca 420

tcacggttgttgtcgacgccgacgagctcaacattgagcagatcaccaagcagctcaaca 480

agctgatccccgtgctcaaagtcgtgcgacttgatgaagagaccaccatcgcccgcgcaa 540

tcatgctggttaaggtctctgcggatagcaccaaccgtccgcagatcgtcgacgccgcga 600

acatcttccgcgcccgagtcgtcgacgtggctccagactctgtggttattgaatccacag 660

gcaccccaggcaagctccgcgcactgcttgatgtgatggaaccattcggaatccgcgaac 720

tgatccaatccggacagattgcactcaaccgcggtccgaagaccatggctccggccaaga 780

tctaagaatttcttcgcgttttttttgcatccatcttgttaacttcgcttattatgggga 840

tcagtttccgggtttcaagggaagcggacacattgtcactatttcatttaacgcagggac 900

ctccaaatcatggctattgaactgctttatgatgctgacgctgacctctccttgatccag 960

ggccgcaaggttgccatcgttggctacggctcccagggccacgcacactcccagaacctc 1020

cgcgattctggcgttgaggttgtcattggtctgcgcgagggctccaagtccgcagagaag 1080

gcaaaggaagcaggcttcgaggtcaagaccaccgctgaggctgcagcttgggctgacgtc 1140

atcatgctcctggctccagacacctcccaggcagaaatcttcaccaacgacatcgagcca 1200

aacctgaacgcaggcgacgcactgctgttcggccacggcctgaacattcacttcgacctg 1260

atcaagccagctgacgacatcatcgttggcatggttgcgccaaagggcccaggccacttg 1320

gttcgccgtcagttcgttgatggcaagggtgttccttgcctcatcgcagtcgaccaggac 1380

ccaaccggaaccgcacaggctctgaccctgtcctacgcagcagcaatcggtggcgcacgc 1440

gcaggcgttatcccaaccaccttcgaagctgagaccgtcaccgacctcttcggcgagcag 1500

gctgttctctgcggtggcaccgaggaactggtcaaggttggcttcgaggttctcaccgaa 1560

gctggctacgagccagagatggcatacttcgaggttcttcacgagctcaagctcatcgtt 1620

gacctcatgttcgaaggtggcatcagcaacatgaactactctgtttctgacaccgctgag 1680

ttcggtggctacctctccggcccacgcgtcatcgatgcagacaccaagtcccgcatgaag 1740

gacatcctgaccgatatccaggacggcaccttcaccaagcgcctcagggtaccgagctcg 1800

aattcgtaat catggtcata gctg 1824

<210> 21

<211> 901

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 21

cccgactttgttaccctttctgagggacttggctgtgttgccatccgcgtcaccaaagcg 60

gaggaagtactgccagccatccaaaaggctcgagagatcaacgaccgcccagtagtcatc 120

gacttcatcgtcggtgaagacgcacaggtatggccaatggtgtctgctggatcatccaac 180

tccgatatccagtacgcactcggattgcgcccattctttgatggtgatgaatctgcagca 240

gaagatcctgccgacattcacgaagccgtcagcgacattgatgccgccgttgaatcgacc 300

gaggcataaggagagacccaagatggctaattctgacgtcacccgccacatcctgtccgt 360

actcgttcaggacgtagacggaatcatttcccgcgtatcaggtatgttcacccgacgcgc 420

attcaacctcgtgtccctcgtgtctgcaaagaccgaaacactcggcatcaaccgcatcac 480

ggttgttgtcgacgccgacgagctcaacattgagcagatcaccaagcagctcaacaagct 540

gatccccgtgctcaaagtcgtgcgacttgatgaagagaccaccatcgcccgcgcaatcat 600

gctggttaaggtctctgcggatagcaccaaccgtccgcagatcgtcgacgccgcgaacat 660

cttccgcgcccgagtcgtcgacgtggctccagactctgtggttattgaatccacaggcac 720

cccaggcaagctccgcgcactgcttgatgtgatggaaccattcggaatccgcgaactgat 780

ccaatccggacagattgcactcaaccgcggtccgaagaccatggctccggccaagatcta 840

agaatttctccgcgtttttttcgcattcatctcgctaacttcgcttattatggggatcag 900

t 901

<210> 22

<211> 901

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 22

cccgactttgttaccctttctgagggacttggctgtgttgccatccgcgtcaccaaagcg 60

gaggaagtactgccagccatccaaaaggctcgagagatcaacgaccgcccagtagtcatc 120

gacttcatcgtcggtgaagacgcacaggtatggccaatggtgtctgctggatcatccaac 180

tccgatatccagtacgcactcggattgcgcccattctttgatggtgatgaatctgcagca 240

gaagatcctgccgacattcacgaagccgtcagcgacattgatgccgccgttgaatcgacc 300

gaggcataaggagagacccaagatggctaattctgacgtcacccgccacatcctgtccgt 360

actcgttcaggacgtagacggaatcatttcccgcgtatcaggtatgttcacccgacgcgc 420

attcaacctcgtgtccctcgtgtctgcaaagaccgaaacactcggcatcaaccgcatcac 480

ggttgttgtcgacgccgacgagctcaacattgagcagatcaccaagcagctcaacaagct 540

gatccccgtgctcaaagtcgtgcgacttgatgaagagaccaccatcgcccgcgcaatcat 600

gctggttaaggtctctgcggatagcaccaaccgtccgcagatcgtcgacgccgcgaacat 660

cttccgcgcccgagtcgtcgacgtggctccagactctgtggttattgaatccacaggcac 720

cccaggcaagctccgcgcactgcttgatgtgatggaaccattcggaatccgcgaactgat 780

ccaatccggacagattgcactcaaccgcggtccgaagaccatggctccggccaagatcta 840

agaatttcttcgcgttttttttgcatccatcttgttaacttcgcttattatggggatcag 900

t 901

<210> 23

<211> 1023

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 23

cgcttattatggggatcagtttcagggtttcaagggaagcactcacattgtcatcaatct 60

tcgcaacaaggacctcggaaaaatggctattgaactgctttatgatgctgacgctgacct 120

ctccttgatccagggccgcaaggttgccatcgttggctacggctcccagggccacgcaca 180

ctcccagaacctccgcgattctggcgttgaggttgtcattggtctgcgcgagggctccaa 240

gtccgcagagaaggcaaaggaagcaggcttcgaggtcaagaccaccgctgaggctgcagc 300

ttgggctgacgtcatcatgctcctggctccagacacctcccaggcagaaatcttcaccaa 360

cgacatcgagccaaacctgaacgcaggcgacgcactgctgttcggccacggcctgaacat 420

tcacttcgacctgatcaagccagctgacgacatcatcgttggcatggttgcgccaaaggg 480

cccaggccacttggttcgccgtcagttcgttgatggcaagggtgttccttgcctcatcgc 540

agtcgaccaggacccaaccggaaccgcacaggctctgaccctgtcctacgcagcagcaat 600

cggtggcgcacgcgcaggcgttatcccaaccaccttcgaagctgagaccgtcaccgacct 660

cttcggcgagcaggctgttctctgcggtggcaccgaggaactggtcaaggttggcttcga 720

ggttctcaccgaagctggctacgagccagagatggcatacttcgaggttcttcacgagct 780

caagctcatcgttgacctcatgttcgaaggtggcatcagcaacatgaactactctgtttc 840

tgacaccgctgagttcggtggctacctctccggcccacgcgtcatcgatgcagacaccaa 900

gtcccgcatgaaggacatcctgaccgatatccaggacggcaccttcaccaagcgcctcat 960

cgcaaacgttgagaacggcaacaccgaacttgagggccttcgtgcttcctacaacaacca 1020

ccc 1023

<210> 24

<211> 1025

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 24

cgcttattatggggatcagtttccgggtttcaagggaagcggacacattgtcactatttc 60

atttaacgcagggacctccaaatcatggctattgaactgctttatgatgctgacgctgac 120

ctctccttgatccagggccgcaaggttgccatcgttggctacggctcccagggccacgca 180

cactcccagaacctccgcgattctggcgttgaggttgtcattggtctgcgcgagggctcc 240

aagtccgcagagaaggcaaaggaagcaggcttcgaggtcaagaccaccgctgaggctgca 300

gcttgggctgacgtcatcatgctcctggctccagacacctcccaggcagaaatcttcacc 360

aacgacatcgagccaaacctgaacgcaggcgacgcactgctgttcggccacggcctgaac 420

attcacttcgacctgatcaagccagctgacgacatcatcgttggcatggttgcgccaaag 480

ggcccaggccacttggttcgccgtcagttcgttgatggcaagggtgttccttgcctcatc 540

gcagtcgaccaggacccaaccggaaccgcacaggctctgaccctgtcctacgcagcagca 600

atcggtggcgcacgcgcaggcgttatcccaaccaccttcgaagctgagaccgtcaccgac 660

ctcttcggcgagcaggctgttctctgcggtggcaccgaggaactggtcaaggttggcttc 720

gaggttctcaccgaagctggctacgagccagagatggcatacttcgaggttcttcacgag 780

ctcaagctcatcgttgacctcatgttcgaaggtggcatcagcaacatgaactactctgtt 840

tctgacaccgctgagttcggtggctacctctccggcccacgcgtcatcgatgcagacacc 900

aagtcccgcatgaaggacatcctgaccgatatccaggacggcaccttcaccaagcgcctc 960

atcgcaaacgttgagaacggcaacaccgaacttgagggccttcgtgcttcctacaacaac 1020

caccc 1025

Claims

1, DNA molecule with the sequence of SEQ ID No.3 in the sequence table.

2. The biomaterial related to the DNA molecule of claim 1, which is any one of the following B1) to B7):

B1) an expression cassette comprising the DNA molecule of claim 1;

B2) a recombinant vector comprising the DNA molecule of claim 1;

B3) a recombinant vector comprising the expression cassette of B1);

B4) a recombinant microorganism comprising the DNA molecule of claim 1;

B5) a recombinant microorganism comprising the expression cassette of B1);

B6) a recombinant microorganism containing the recombinant vector of B2);

B7) a recombinant microorganism containing the recombinant vector of B3).

3. Use of the DNA molecule of claim 1 as a promoter.

4. Use of the DNA molecule of claim 1 or the biomaterial of claim 2 for the production of amino acids.

5. Use according to claim 4, characterized in that: the amino acid is lysine, glutamic acid or valine.

6. A method of producing an amino acid comprising: introducing the DNA molecule of claim 1 into a biological cell capable of synthesizing the desired amino acid, such that the DNA molecule of claim 1 drives the expression of genes in the synthetic pathway of the desired amino acid in the biological cell, thereby obtaining a recombinant biological cell; culturing the recombinant biological cells to obtain target amino acid;

the biological cell is corynebacterium glutamicum.

7. The method of claim 6, wherein: the target amino acid is lysine, glutamic acid or valine.